Functional characterization of neuropeptide 26RFa receptors GPR103A and GPR103B in zebrafish, Danio rerio

Wang, Weiwei; Jiang, Chaohui; Xu, Yue; Ma, Qiang; Yang, Jingwen; Shi, Ying; Zhou, Naiming

doi:10.1016/j.cellsig.2020.109677

Cited by 1 publication

(1 citation statement)

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“…The data were used One-or Two-way ANOVA with Tukey's post hoc test to analyze data for continuous variables with normal distributions. In this study, no pre-determined sample size calculation was applied, and the sample sizes were based on previous studies (Soto & Trejo, 2010;Wang et al, 2020). Blinding was not performed and test for outliers was not conducted.…”

Section: Discussionmentioning

confidence: 99%

N‐glycosylation of the human neuropeptide QRFP receptor (QRFPR) is essential for ligand binding and receptor activation

Wang

Yan

Shi

et al. 2021

Journal of Neurochemistry

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The newly identified pyroglutamylated RFamide peptide (QRFP) signaling system has been shown to be implicated in regulating a variety of physiological processes. G-protein-coupled receptors (GPCRs) are preferentially N-glycosylated on extracellular domains. The human QRFP receptor QRFPR (GPR103) possesses three Nglycosylation consensus sites, two located on the N-terminal domain (N5 and N19) and one on the first extracellular loop (ECL1) (N106); however, to date, their role in QRFPR expression and signaling has not been established. Here, we combined mutants with glutamine substitution of the critical asparagines of the consensus sites with glycosidase PNGase F and N-glycosylation inhibitor tunicamycin to study the effect of N-glycosylation in the regulation of QRFPR cell surface expression and signaling. Western blot analysis performed with site-directed mutagenesis revealed that two asparagines at N19 in the N-terminus and N106 in ECL1, but not N5 in the N-terminus, served as sites for N-glycosylation. Treatment with PNGase F and tunicamycin resulted in a reduction in both two-protein species, ~43 kDa and ~85 kDa in size, by 2-4 kDa. Analysis with confocal microscopy and quantitative ELISA showed that N-glycosylation of QRFPR is not essentially required for targeting the cell membrane. However, further binding assay and functional assays demonstrated that removal of N-glycosylation sequons or treatment with tunicamycin led to significant impairments in the interaction of receptor with QRFP26 and downstream signaling. Thus, our findings suggest that for the human QRFP receptor (QRFPR), N-glycosylation is not important for cell surface expression but is a pre-requisite for ligand binding and receptor activation.

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Section: Discussionmentioning

confidence: 99%