Chemokine CXCL12 induces binding of ferritin heavy chain to the chemokine receptor CXCR4, alters CXCR4 signaling, and induces phosphorylation and nuclear translocation of ferritin heavy chain.
Chemokine receptor-initiated signaling plays critical roles in cell differentiation, proliferation, and migration. However, the regulation of chemokine receptor signaling under physiological and pathological conditions is not fully understood. In the present study, we demonstrate that the CXC chemokine receptor 4 (CXCR4) formed a complex with ferritin heavy chain (FHC) in a ligand-dependent manner. Our in vitro binding assays revealed that purified FHC associated with both the glutathione S-transferase-conjugated N-terminal and C-terminal domains of CXCR4, thereby suggesting the presence of more than one FHC binding site in the protein sequence of CXCR4. Using confocal microscopy, we observed that stimulation with CXCL12, the receptor ligand, induced colocalization of the internalized CXCR4 with FHC into internal vesicles. Furthermore, after CXCL12 treatment, FHC underwent time-dependent nuclear translocation and phosphorylation at serine residues. By contrast, a mutant form of FHC in which serine 178 was replaced by alanine (S178A) failed to undergo phosphorylation, suggesting that serine 178 is the major phosphorylation site. Compared with the wild type FHC, the FHC-S178A mutant exhibited reduced association with CXCR4 and constitutive nuclear translocation. We also found that CXCR4-mediated extracellular signal-regulated kinase 1/2 (ERK1/2) activation and chemotaxis were inhibited by overexpression of wild type FHC but not FHC-S178A mutant, and were prolonged by FHC knockdown. In addition to CXCR4, other chemokine receptor-initiated signaling appeared to be similarly regulated by FHC, because CXCR2-mediated ERK1/2 activation was also inhibited by FHC overexpression and prolonged by FHC knockdown. Altogether, our data provide strong evidence for an important role of FHC in chemokine receptor signaling and receptor-mediated cell migration.
Li
Runsheng
R
Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and Shanghai Jiaotong University School of Medicine, Shanghai 200025, People's Republic of China.
Luo
Cherry
C
Mines
Marjelo
M
Zhang
Jingwu
J
Fan
Guo-Huang
GH
D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D001052:Apoferritins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D054377:Chemokine CXCL12; D019743:Chemokines, CXC / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D019169:Jurkat Cells; D020935:MAP Kinase Signaling System / Q000187:drug effects; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D019718:Receptors, CXCR4 / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection
C515255:CXCL12 protein, human; D054377:Chemokine CXCL12; D019743:Chemokines, CXC; D034741:RNA, Small Interfering; D019718:Receptors, CXCR4; D011994:Recombinant Proteins; D001052:Apoferritins; D048049:Extracellular Signal-Regulated MAP Kinases