Role of Tyrosine Phosphorylation in Ligand-independent Sequestration of CXCR4 in Human Primary Monocytes-Macrophages (2001)

The chemokine stromal cell-derived factor (SDF)-1 and its receptor, CXCR4, play important roles in human immunodeficiency virus type 1 (HIV-1) pathophysiology, leukocyte trafficking, inflammation, hematopoiesis, embryogenesis, angiogenesis, and cancer metastasis. The effects of cytokines on the regulation of CXCR4 function were investigated in human primary monocytes-macrophages. The expression of functional CXCR4 on the cell surface was demonstrated by the detection of ligand-induced Ca2+ mobilization, chemotaxis, and ligand-induced receptor endocytosis. Surface CXCR4 expression was down-regulated by cytokines interleukin-4 (IL-4), IL-13, and granulocyte-macrophage colony-stimulating factor (GM-CSF) and up-regulated by IL-10 and transforming growth factor-β1. Down-regulation was mediated post-translationally, in the absence of protein degradation, through an endocytotic mechanism. In contrast to SDF-1α-induced CXCR4 endocytosis, cytokine-induced endocytosis of this receptor was independent of actin filament polymerization. GM-CSF increased the expression of G protein-coupled receptor kinase 3 (GRK3), β-arrestin-1, Pyk2, and focal adhesion kinase (FAK). Cytokine treatment also increased the total and tyrosine-specific phosphorylation of CXCR4 as well as the phosphorylation of FAK on tyrosine 397. It also induced the formation of GRK3·CXCR4 or FAK·CXCR4 complexes. Infection of macrophages by primary R5X4 and X4 isolates of HIV-1 was inhibited by IL-4, IL-13, and GM-CSF, an effect that was associated with down-regulation of surface CXCR4 expression. These data indicate that ligand-dependent and ligand-independent endocytoses of CXCR4 are mediated by different mechanisms. Cytokine-induced endocytosis of chemokine receptors may be of therapeutic value in HIV-1 infection, inflammation, tumor metastasis, and defective hematopoiesis. The chemokine stromal cell-derived factor (SDF)-1 and its receptor, CXCR4, play important roles in human immunodeficiency virus type 1 (HIV-1) pathophysiology, leukocyte trafficking, inflammation, hematopoiesis, embryogenesis, angiogenesis, and cancer metastasis. The effects of cytokines on the regulation of CXCR4 function were investigated in human primary monocytes-macrophages. The expression of functional CXCR4 on the cell surface was demonstrated by the detection of ligand-induced Ca2+ mobilization, chemotaxis, and ligand-induced receptor endocytosis. Surface CXCR4 expression was down-regulated by cytokines interleukin-4 (IL-4), IL-13, and granulocyte-macrophage colony-stimulating factor (GM-CSF) and up-regulated by IL-10 and transforming growth factor-β1. Down-regulation was mediated post-translationally, in the absence of protein degradation, through an endocytotic mechanism. In contrast to SDF-1α-induced CXCR4 endocytosis, cytokine-induced endocytosis of this receptor was independent of actin filament polymerization. GM-CSF increased the expression of G protein-coupled receptor kinase 3 (GRK3), β-arrestin-1, Pyk2, and focal adhesion kinase (FAK). Cytokine treatment also increased the total and tyrosine-specific phosphorylation of CXCR4 as well as the phosphorylation of FAK on tyrosine 397. It also induced the formation of GRK3·CXCR4 or FAK·CXCR4 complexes. Infection of macrophages by primary R5X4 and X4 isolates of HIV-1 was inhibited by IL-4, IL-13, and GM-CSF, an effect that was associated with down-regulation of surface CXCR4 expression. These data indicate that ligand-dependent and ligand-independent endocytoses of CXCR4 are mediated by different mechanisms. Cytokine-induced endocytosis of chemokine receptors may be of therapeutic value in HIV-1 infection, inflammation, tumor metastasis, and defective hematopoiesis. human immunodeficiency virus type 1 monocyte-derived macrophage G protein-coupled receptor GPCR kinase stromal cell-derived factor focal adhesion kinase cytosolic free Ca2+ concentration phosphatidylcholine-specific phospholipase C phosphatidylinositol protein kinase C granulocyte-macrophage colony-stimulating factor macrophage/colony-stimulating factor regulated on activation normal T cell expressed and secreted signal transducers and activators of transcription interferon γ transforming growth factor-β monoclonal antibody phosphatidylinositol 3-kinase Janus kinase phycoerythrin Macrophages and T lymphocytes are the major targets of HIV-11 infection and constitute the productive viral reservoirs (1Perrin-Tricaud C. Davoust J. Jones I.M. Virology. 1999; 255: 20-25Crossref PubMed Scopus (30) Google Scholar, 2Ott D.E. Coren L.V. 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The cells, were in macrophage in and cells were in with and were by and IL-4, IL-13, and antibody to were from and GM-CSF was from IL-4, IL-13, and were from and were from to were from indicated were from were with the to CXCR4 and then subjected to as previously (11Wang J. Guan E. Roderiquez G. Norcross M.A. J. Immunol. 1999; 163: 5763-5769PubMed Google Scholar). Primary R5X4 virus as well as primary X4 virus were from the of and for with indicated cytokines or were to virus for the cells were with and then in for an were and for with an was with a were with and the cytosolic free Ca2+ concentration was by of and with a as previously (11Wang J. Guan E. Roderiquez G. Norcross M.A. J. Immunol. 1999; 163: 5763-5769PubMed Google Scholar). Macrophages were and in of the cell were in the of with a of The or not with was in the for in a the of cells that through the were with a for In cells were with the to CXCR4 or with an and for into the was from macrophages with the use of The was for the of in the of of and of total were subjected to with the and then subjected to for with a of and were by on a and to Macrophages on were and with to the of CXCR4 and then with to In cells were with surface and was with an from and was and was by with or with by were and as previously J. Guan E. G. Norcross M.A. J. Immunol. PubMed Scopus Google Scholar). the of were for with protein with primary and protein cell or were by on to and as previously J. Guan E. G. Norcross M.A. J. Immunol. PubMed Scopus Google Scholar). in were with cytokines and then by for with 1 of in in the absence of were then and subjected to as previously Ferguson S.S. J. M.G. J. 1997; PubMed Scopus Google Scholar). In the cells were in for 1 and then for of the were for with protein for with of to the of CXCR4 the of protein and for an the were with and were in by for The were then subjected to on a to by The of X4 viruses to either that the CXCR4 expressed on cells is defective for infection or that of are on but not on T the functional of CXCR4 in Ca2+ mobilization, modulation of CXCR4 and induced by The surface expression of CXCR4 was down-regulated by as well as by in as as was in the T cell and that not with the interaction of with CXCR4, cells with to the from the receptor with the The expression of CXCR4 was not by treatment not that not with the interaction of the a in CXCR4 A. N. K. M. J. Virol. 1999; PubMed Google Scholar, A. N. M. M. J. Biol. 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We investigated the effect of cytokines on the expression of CXCR4 is mediated the of transcription or The of CXCR4 and protein in was not by to cytokines and that down-regulation of surface CXCR4 expression by cytokines is not mediated the of transcription and induce endocytosis of a that has been implicated in receptor regulation by sequestration of receptors in or by of receptors in that cytokines were shown to CXCR4 expression on the surface of macrophages the of CXCR4 or total the effects of on CXCR4 endocytosis. were with to CXCR4, and with a In cells, CXCR4 was the cell surface In in cells with GM-CSF, CXCR4 was to the of the to the a that that of CXCR4 in cells with or N. M. J. 1998; PubMed Google Scholar). and data thus indicate that CXCR4 is in the in than in of macrophages with The is with the A. Rev. Biol. 1998; PubMed Scopus Google Scholar). The of actin may be for the of the and the G. J. Biol. 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The also increased the expression of CXCR4 on the cell but not the down-regulation of surface CXCR4 expression induced by IL-4, IL-13, or GM-CSF not that also not to this of of GPCRs is to be for ligand-induced endocytosis T. M. Full Full PubMed Scopus Google Scholar). CXCR4 is phosphorylated during cytokine-induced endocytosis in phosphorylation CXCR4 a of phosphorylation in cells in of cells to or GM-CSF in a in the of CXCR4 that cytokine-induced endocytosis of CXCR4 was by CXCR4 tyrosine phosphorylation during this that to CXCR4 from of but not from of cells These results thus that tyrosine phosphorylation of CXCR4 to the cytokine-induced endocytosis of this phosphorylation by and the of the phosphorylated receptor with β-arrestin are important in and sequestration of GPCRs Y. M. J.D. Lefkowitz R.J. U. S. A. 1997; PubMed Scopus Google Scholar, A.M. A. S. A. F. 1998; PubMed Scopus Google Scholar, M. M. Proudfoot A.E. H. J. Biol. 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Roderiquez G. Oravecz T. Norcross M.A. J. Virol. 1998; 72: 7642-7647Crossref PubMed Google GM-CSF both CCR5 expression and R5 virus infection. cytokines effects on X4 virus infection, with the cytokines to Infection of by the primary X4 virus or the R5X4 virus was inhibited by IL-4, IL-13, or GM-CSF that and both the CCR5 and the CXCR4 of viral GM-CSF inhibited the infection of macrophages by R5X4 virus its effect on CCR5 expression (12Wang J. Roderiquez G. Oravecz T. Norcross M.A. J. Virol. 1998; 72: 7642-7647Crossref PubMed Google Scholar). The X4 virus to infect in the of was with an R5 cells not We and have previously effects of as on CCR5 expression by macrophages and on infection of cells by R5 or R5X4 HIV-1 isolates (12Wang J. Roderiquez G. Oravecz T. Norcross M.A. J. Virol. 1998; 72: 7642-7647Crossref PubMed Google Scholar, J. Immunol. 2000; PubMed Scopus Google Scholar). We have by the effects of cytokines on CXCR4 function in macrophages and on infection of cells by primary We that CXCR4 expressed on the surface of to its by an in and by both and receptor endocytosis. The cytokines IL-4, IL-13, and GM-CSF inhibited infection of macrophages by primary X4 and GM-CSF was shown to cell and Ca2+ in to Cytokine of was associated with down-regulation of surface expression of The function of CXCR4 on macrophages as a for HIV-1 infection has been The that X4 viruses do not replicate in to the of and virus The resistance of macrophages to viruses is not to a lack of CXCR4 expression or to in CXCR4 as ligand-induced Ca2+ and receptor endocytosis, as shown and in The that primary X4 viruses are to infect cells also that CXCR4 is to virus The down-regulation of surface CXCR4 expression by IL-4, IL-13, or GM-CSF in the is not to an in the of the CXCR4 or as demonstrated by antibody The expression of CXCR4 on the cell surface was also shown not to from cytokine-induced of CXCR4 transcription or that this is mediated by an in receptor endocytosis. 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