Original Article

Chemerin C9 peptide induces receptor internalization through a clathrin-independent pathway

Authors: Jun-xian Zhou, Dan Liao, Shuo Zhang, Ni Cheng, Hui-qiong He, Richard D Ye
DOI: 10.1038/aps.2013.198

Abstract

Jun-xian ZHOU1, Dan LIAO1, Shuo ZHANG1, Ni CHENG2, Hui-qiong HE1, Richard D YE1, 2, *
1School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China; 2Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL 60612, USA

Aim: The chemerin receptor CMKLR1 is one type of G protein-coupled receptors abundant in monocyte-derived dendritic cells and macrophages, which plays a key role in the entry of a subset of immunodeficiency viruses including HIV/SIV into lymphocytes and macrophages. The aim of this work was to investigate how CMKLR1 was internalized and whether its internalization affected cell signaling in vitro.
Methods: Rat basophilic leukemia RBL-2H3 cells, HEK 293 cells, and HeLa cells were used. CMKLR1 internalization was visualized by confocal microscopy imaging or using a FACScan flow cytometer. Six potential phosphorylation sites (Ser337, Ser343, Thr352, Ser344, Ser347, and Ser350) in CMKLR1 were substituted with alanine using site-directed mutagenesis. Heterologous expression of wild type and mutant CMKLR1 allowed for functional characterization of endocytosis, Ca2+ flux and extracellular signal-regulated kinase (ERK) phosphorylation.

Results: Chemerin and the chemerin-derived nonapeptide (C9) induced dose-dependent loss of cell surface CMKLR1-GFP fusion protein and increased its intracellular accumulation in HEK 293 cells and RBL-2H3 cells stably expressing CMKLR1. Up to 90% of CMKLR1 was internalized after treatment with C9 (1 ┬Ámol/L). By using different agents, it was demonstrated that clathrin-independent mechanism was involved in CMKLR1 internalization. Mutations in Ser343 for G protein-coupled receptor kinase phosphorylation and in Ser347 for PKC phosphorylation abrogated CMKLR1 internalization. Loss of CMKLR1 internalization partially enhanced the receptor signaling, as shown by increased Ca2+ flux and a shorter latency to peak level of ERK phosphorylation.

Conclusion: CMKLR1 internalization occurs in a clathrin-independent manner, which negatively regulated the receptor-mediated Ca2+ flux and ERK phosphorylation.


Keywords: chemerin; CMKLR1; GPCR; receptor trafficking; receptor internalization; endocytosis; calcium flux; ERK phosphorylation

This work was supported, in part, by the National Natural Science Foundation of China (Grant 31270941), the National Basic Research Program of China (973 Program Grant 2012CB518000), the Specialized Research Fund for the Doctoral Program of Higher Education of China (Grant 20120073110069), and the US National Institutes of Health (Grant R01 AI 033503).
* To whom correspondence should be addressed.
E-mail yedequan@sjtu.edu.cn
Received 2013-08-17 Accepted 2013-12-08
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