RESUMO
The chemokine CCL20 is broadly produced by endothelial cells in the liver, the lung, in lymph nodes and mucosal lymphoid tissues, and recruits CCR6 expressing leukocytes, particularly dendritic cells, mature B cells, and subpopulations of T cells. How CCL20 is systemically scavenged is currently unknown. Here, we identify that fluorescently labeled human and mouse CCL20 are efficiently taken-up by the atypical chemokine receptor ACKR4. CCL20 shares ACKR4 with the homeostatic chemokines CCL19, CCL21, and CCL25, although with a lower affinity. We demonstrate that all 4 human chemokines recruit ß-arrestin1 and ß-arrestin2 to human ACKR4. Similarly, mouse CCL19, CCL21, and CCL25 equally activate the human receptor. Interestingly, at the same chemokine concentration, mouse CCL20 did not recruit ß-arrestins to human ACKR4. Further cross-species analysis suggests that human ACKR4 preferentially takes-up human CCL20, whereas mouse ACKR4 similarly internalizes mouse and human CCL20. Furthermore, we engineered a fluorescently labeled chimeric chemokine consisting of the N-terminus of mouse CCL25 and the body of mouse CCL19, termed CCL25_19, which interacts with and is taken-up by human and mouse ACKR4.
Assuntos
Quimiocina CCL19/metabolismo , Quimiocina CCL20/metabolismo , Quimiocina CCL21/metabolismo , Quimiocinas CC/metabolismo , Receptores CCR/metabolismo , beta-Arrestinas/genética , Sequência de Aminoácidos , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Sítios de Ligação , Linhagem Celular , Quimiocina CCL19/química , Quimiocina CCL19/genética , Quimiocina CCL20/química , Quimiocina CCL20/genética , Quimiocina CCL21/química , Quimiocina CCL21/genética , Quimiocinas CC/química , Quimiocinas CC/genética , Células HEK293 , Células HeLa , Humanos , Ligantes , Camundongos , Proteínas Mutantes Quiméricas/química , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Secundária de Proteína , Receptores CCR/química , Receptores CCR/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Transfecção , beta-Arrestinas/metabolismoRESUMO
The chemokine receptor CCR7 plays a pivotal role in health and disease. In particular, CCR7 controls homing of antigen-bearing dendritic cells and T cells to lymph nodes, where adaptive immune responses are initiated. However, CCR7 also guides T cells to inflamed synovium and thereby contributes to rheumatoid arthritis and promotes cancer cell migration and metastasis formation. Nanobodies have recently emerged as versatile tools to study G-protein-coupled receptor functions and are being tested in diagnostics and therapeutics. In this study, we designed a strategy to engineer novel nanobodies recognizing human CCR7. We generated a nanobody library based on a solved crystal structure of the nanobody Nb80 recognizing the ß2-adrenergic receptor (ß2AR) and by specifically randomizing two segments within complementarity determining region 1 (CDR1) and CDR3 of Nb80 known to interact with ß2AR. We fused the nanobody library to one half of split-YFP in order to identify individual nanobody clones interacting with CCR7 fused to the other half of split-YFP using bimolecular fluorescence complementation. We present three novel nanobodies, termed Nb1, Nb5, and Nb38, that recognize human CCR7 without interfering with G-protein-coupling and downstream signaling. Moreover, we were able to follow CCR7 trafficking upon CCL19 triggering using Nb1, Nb5, and Nb38.