title: Chemerin C9 peptide induces receptor internalization through a clathrin-independent pathway.
abstract: OBJECTIVE
  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, Ca(2+) 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 μmol/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 Ca(2+) flux and a shorter latency to peak level of ERK phosphorylation.
  
  
  CONCLUSIONS
  CMKLR1 internalization occurs in a clathrin-independent manner, which negatively regulated the receptor-mediated Ca(2+) flux and ERK phosphorylation.
authors.affiliation: School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Jun-xian
authors.initials: JX
authors.lastname: Zhou
authors.affiliation: School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Liao
authors.affiliation: School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Shuo
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Department of Pharmacology, University of Illinois College of Medicine, Chicago, Illinois 60612, USA.
authors.forename: Ni
authors.initials: N
authors.lastname: Cheng
authors.affiliation: School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Hui-qiong
authors.initials: HQ
authors.lastname: He
authors.affiliation: 1] School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China [2] Department of Pharmacology, University of Illinois College of Medicine, Chicago, Illinois 60612, USA.
authors.forename: Richard D
authors.initials: RD
authors.lastname: Ye
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D002966:Clathrin / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D015471:Leukemia, Basophilic, Acute / Q000378:metabolism; D010766:Phosphorylation / Q000502:physiology; D051381:Rats; D019707:Receptors, Chemokine / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*
chemical_list: C102741:CMKLR1 protein, human; D002966:Clathrin; D019707:Receptors, Chemokine; D043562:Receptors, G-Protein-Coupled; D049452:Green Fluorescent Proteins; D048049:Extracellular Signal-Regulated MAP Kinases; D002118:Calcium