ABSTRACT
The chemokine receptor CCR-7 is expressed in T, NK, and dendritic cells in a time-ordered and stimulus-dependent manner. Thorough analyses of the pharmacological profiles of the recombinant ligands for CCR-7, MIP-3beta/ELC/CK-beta 11, and SLC/Exodus-2/TCA4/6C-kine, using CCR-7-expressing HEK-293E transfectants determine that ligands both bind with a K(d) in the 100 pM range-10- to 100-fold greater affinities than published K(d) values. High-affinity binding of each ligand is associated with rapid mobilization of intracellular calcium and cell migration as predicted for chemokine GPCRs, and in keeping with more recent evidence, robust activation of mitogen-activated protein kinase (MAPK).
Subject(s)
Calcium/metabolism , Chemokines, CC/metabolism , Receptors, Chemokine/physiology , Signal Transduction/physiology , Cell Line , Cell Membrane/physiology , Chemokine CCL19 , Chemokine CCL21 , Chemokines, CC/pharmacology , Chemotaxis , Cloning, Molecular , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Humans , Kidney , Kinetics , Ligands , Mitogen-Activated Protein Kinases/metabolism , Phosphorylation , Radioligand Assay , Receptors, CCR7 , Receptors, Chemokine/genetics , Receptors, Chemokine/metabolism , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Signal Transduction/drug effects , TransfectionABSTRACT
The CC chemokine macrophage inflammatory protein-3alpha (MIP-3alpha) is the product of recent electronic cloning efforts, however, little characterization of its spectrum of biological effects has been undertaken. Human eosinophils exhibited pertussis-toxin-sensitive migration in response to human recombinant (hr)MIP-3alpha. Messenger RNA for the MIP-3alpha receptor, CCR-6, and low levels of surface expression were demonstrated by reverse transcriptase-polymerase chain reaction and FACS analysis. Analyses of cell signaling revealed dose-dependent increases in intracellular calcium mobilization, calcium transients that were, however, greatly reduced when compared with MCP-3-induced responses. Further investigations of MIP-3alpha-induced signal transduction revealed time- and dose-dependent, partially pertussis toxin-dependent, increases in phosphorylation of the p42/p44 mitogen-activated protein kinases (MAPK) that occurred at 10- to 100-fold lower concentrations, and that were linked to a phosphoinositide 3-kinase pathway. These results suggest that MIP-3alpha can regulate multiple, parallel signal transduction pathways in eosinophils, and suggest that MAPK activation by MIP-3alpha in eosinophils is a significant signaling pathway for migration induction.
