Phosphorylation by a G protein-coupled kinase inhibits signaling and promotes internalization of the monocyte chemoattractant protein-1 receptor. Critical role of carboxyl-tail serines/threonines in receptor function.
Monocyte chemoattractant protein-1 (MCP-1) is a member of the chemokine family of chemotactic cytokines and signals via activation of a G protein-coupled seven-transmembrane domain receptor to mediate chemotaxis. Monocyte activation is limited by desensitization and internalization of the MCP-1R, but these mechanisms are not well understood. In this study, we show that the type B MCP-1R (MCP-1RB/CCR2B) is rapidly phosphorylated and internalized in response to nanomolar concentrations of MCP-1. Co-expression of CCR2B in Xenopus oocytes with beta-adrenergic receptor kinase 2 (beta ark2), but not beta ark1 or rhodopsin kinase, specifically blocked receptor activation by MCP-1. Mutation of serine (Ser) and threonine (Thr) residues in the terminal carboxyl-tail of the receptor, which are potential targets of beta ark-mediated phosphorylation, prevented inhibition of receptor activation by beta ark2 in microinjected oocytes. Finally, a construct in which multiple Ser and Thr residues in the carboxyl-tail were changed to alanine significantly prolonged the agonist-dependent intracellular calcium flux and inhibited receptor internalization in transfected human embryonic kidney (HEK)-293 cells. These studies demonstrate that phosphorylation of Ser and Thr residues in the carboxyl-tail of CCR2B mediates receptor desensitization and internalization and may serve to limit the chemotactic response of leukocytes to MCP-1 and related chemokines.
Franci
C
C
Gladstone Institute of Cardiovascular Disease, University of California, San Francisco 94143, USA.
Gosling
J
J
Tsou
C L
CL
Coughlin
S R
SR
Charo
I F
IF
D000595:Amino Acid Sequence; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D054390:Receptors, CCR2; D019707:Receptors, Chemokine; D018121:Receptors, Cytokine / Q000737:chemistry / Q000502:physiology*; D011993:Recombinant Fusion Proteins; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D013912:Threonine / Q000737:chemistry; D014407:Tumor Cells, Cultured; D051552:beta-Adrenergic Receptor Kinases
C515334:CCR2 protein, human; D054390:Receptors, CCR2; D019707:Receptors, Chemokine; D018121:Receptors, Cytokine; D011993:Recombinant Fusion Proteins; D013912:Threonine; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D019204:GTP-Binding Proteins