title: Differential desensitization, receptor phosphorylation, beta-arrestin recruitment, and ERK1/2 activation by the two endogenous ligands for the CC chemokine receptor 7.
abstract: Many members of the chemokine receptor family of G protein-coupled receptors utilize multiple endogenous ligands. However, differences between the signaling properties of multiple chemokines through a single receptor have yet to be well characterized. In this study we investigated the early signaling events of CCR7 initiated by its two endogenous ligands, CCL19 and CCL21. Both CCL19 and CCL21 induce G protein activation and calcium mobilization with equal potency. However, only activation by CCL19, not CCL21, promotes robust desensitization of endogenous CCR7 in the human T cell lymphoma cell line H9. Desensitization occurs through the induction of receptor phosphorylation and beta-arrestin recruitment (shown in HEK293 cells expressing CCR7-FLAG). The sites of CCL19-induced phosphorylation were mapped by mutating to alanines the serines and threonines found within kinase phosphorylation consensus sequences in the carboxyl terminus of CCR7. A cluster of sites, including Thr-373-376 and Ser-378 is important for CCL19-mediated phosphorylation of the receptor, whereas residues serine 356, 357, 364, and 365 are important for basal receptor phosphorylation by protein kinase C. Activation of CCR7 by both ligands leads to signaling to the ERK1/2 mitogen-activated protein kinase pathway. However, CCL19 promotes 4-fold more ERK1/2 phosphorylation than does CCL21. The mechanism by which CCL19 activates ERK1/2 was determined to be beta-arrestin-dependent, because it is reduced both by depletion of beta-arrestin-2 with small interfering RNA and by elimination of the phosphorylation sites in the tail of the receptor. Taken together, these findings demonstrate that CCL19 and CCL21 place CCR7 in functionally distinct conformations that are independent of their G protein-coupling potency: one that allows the efficient desensitization of the receptor and activation of ERK1/2, and another that is impaired in these functions.
authors.affiliation: Department of Exploratory Discovery, Neurocrine Biosciences Inc., San Diego, California 92121, USA. tkohout@neurocrine.com
authors.forename: Trudy A
authors.initials: TA
authors.lastname: Kohout
authors.forename: Shelby L
authors.initials: SL
authors.lastname: Nicholas
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Perry
authors.forename: Greg
authors.initials: G
authors.lastname: Reinhart
authors.forename: Sachiko
authors.initials: S
authors.lastname: Junger
authors.forename: R Scott
authors.initials: RS
authors.lastname: Struthers
mesh_terms: D019390:Arrestins / Q000378:metabolism*; D045744:Cell Line, Tumor; D054415:Chemokine CCL19; D054421:Chemokine CCL21; D019742:Chemokines, CC / Q000378:metabolism; D006801:Humans; D008024:Ligands; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation; D011487:Protein Conformation; D054400:Receptors, CCR7; D019707:Receptors, Chemokine / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
chemical_list: C000605254:ARRB2 protein, human; D019390:Arrestins; C515460:CCL19 protein, human; C515478:CCL21 protein, human; C515370:CCR7 protein, human; D054415:Chemokine CCL19; D054421:Chemokine CCL21; D019742:Chemokines, CC; D008024:Ligands; D054400:Receptors, CCR7; D019707:Receptors, Chemokine; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases