Inhibition of dynamin prevents CCL2-mediated endocytosis of CCR2 and activation of ERK1/2.
The magnitude and duration of G protein-coupled receptor (GPCR) signals are regulated through desensitization mechanisms. In leukocytes, ligand binding to chemokine receptors leads to Ca2+ mobilization and ERK activation through pertussis toxin-sensitive G proteins, as well as to phosphorylation of the GPCR. After interaction with the endocytic machinery (clathrin, adaptin), the adaptor beta-arrestin recognizes the phosphorylated GPCR tail and quenches signaling to receptors. The molecular mechanisms that lead to receptor endocytosis are not universal amongst the GPCR, however, and the precise spatial and temporal events in the internalization of the CCR2 chemokine receptor remain unknown. Here we show that after ligand binding, CCR2 internalizes rapidly and reaches early endosomes, and later, lysosomes. Knockdown of clathrin by RNA interference impairs CCR2 internalization, as does treatment with the dynamin inhibitor, dynasore. Our results show that CCR2 internalization uses a combination of clathrin-dependent and -independent pathways, as observed for other chemokine receptors. Moreover, the use of dynasore allowed us to confirm the existence of a dynamin-sensitive element that regulates ERK1/2 activation. Our results indicate additional complexity in the link between receptor internalization and cell signaling.
García Lopez
M A
MA
Department of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, Darwin 3, 28049 Madrid, Spain.
Aguado Martínez
A
A
Lamaze
C
C
Martínez-A
C
C
Fischer
T
T
D019390:Arrestins / Q000378:metabolism; D002460:Cell Line; D018932:Chemokine CCL2 / Q000378:metabolism*; D002966:Clathrin / Q000378:metabolism; D034281:Dynamins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004705:Endocytosis; D004789:Enzyme Activation; D006801:Humans; D006835:Hydrazones / Q000494:pharmacology; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D054390:Receptors, CCR2 / Q000378:metabolism*; D000071557:beta-Arrestins
D019390:Arrestins; C111835:CCL2 protein, human; C515334:CCR2 protein, human; D018932:Chemokine CCL2; D002966:Clathrin; D006835:Hydrazones; C511794:N'-(3,4-dihydroxybenzylidene)-3-hydroxy-2-naphthahydrazide; D054390:Receptors, CCR2; D000071557:beta-Arrestins; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D034281:Dynamins