Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation.
IL-8 (or CXCL8) activates the receptors CXCR1 (IL-8RA) and CXCR2 (IL-8RB) to induce chemotaxis in leukocytes, but only CXCR1 mediates cytotoxic and cross-regulatory signals. This may be due to the rapid internalization of CXCR2. To investigate the roles of the intracellular domains in receptor regulation, wild-type, chimeric, phosphorylation-deficient, and cytoplasmic tail (C-tail) deletion mutants of both receptors were expressed in RBL-2H3 cells and studied for cellular activation, receptor phosphorylation, desensitization, and internalization. All but one chimeric receptor bound IL-8 and mediated signal transduction, chemotaxis, and exocytosis. Upon IL-8 activation, the chimeric receptors underwent receptor phosphorylation and desensitization. One was resistant to internalization, yet it mediated normal levels of beta-arrestin 2 (beta arr-2) translocation. The lack of internalization by this receptor may be due to its reduced association with beta arr-2 and the adaptor protein-2 beta. The C-tail-deleted and phosphorylation-deficient receptors were resistant to receptor phosphorylation, desensitization, arrestin translocation, and internalization. They also mediated greater phosphoinositide hydrolysis and exocytosis and sustained Ca(2+) mobilization, but diminished chemotaxis. These data indicate that phosphorylation of the C-tails of CXCR1 and CXCR2 are required for arrestin translocation and internalization, but are not sufficient to explain the rapid internalization of CXCR2 relative to CXCR1. The data also show that receptor internalization is not required for chemotaxis. The lack of receptor phosphorylation was correlated with greater signal transduction but diminished chemotaxis, indicating that second messenger production, not receptor internalization, negatively regulates chemotaxis.
Richardson
Ricardo M
RM
Department of Biochemistry, Meharry Medical College, Nashville, TN 37214, USA. mrrichardson@mmc.edu
Marjoram
Robin J
RJ
Barak
Larry S
LS
Snyderman
Ralph
R
D033962:Adaptor Protein Complex 2 / Q000378:metabolism; D033966:Adaptor Protein Complex beta Subunits / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D019390:Arrestins / Q000235:genetics / Q000378:metabolism; D002634:Chemotaxis, Leukocyte / Q000276:immunology*; D003593:Cytoplasm / Q000235:genetics / Q000276:immunology*; D034284:Dynamin I / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D016209:Interleukin-8 / Q000494:pharmacology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D010766:Phosphorylation; D051381:Rats; D023062:Receptors, Interleukin-8A / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D023063:Receptors, Interleukin-8B / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D019061:src-Family Kinases / Q000378:metabolism
D033962:Adaptor Protein Complex 2; D033966:Adaptor Protein Complex beta Subunits; C000605257:Arrb2 protein, rat; D019390:Arrestins; D016209:Interleukin-8; D010446:Peptide Fragments; D023062:Receptors, Interleukin-8A; D023063:Receptors, Interleukin-8B; D011993:Recombinant Fusion Proteins; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D019061:src-Family Kinases; D034284:Dynamin I; D020558:GTP Phosphohydrolases