β-Arrestins Regulate Interleukin-8-induced CXCR1 Internalization

Abstract

The functional role of neutrophils during acute inflammatory responses is regulated by two high affinity interleukin-8 receptors (CXCR1 and CXCR2) that are rapidly desensitized and internalized upon binding their cognate chemokine ligands. The efficient re-expression of CXCR1 on the surface of neutrophils following agonist-induced internalization suggests that CXCR1 surface receptor turnover may involve regulatory pathways and intracellular factors similar to those regulating β2-adrenergic receptor internalization and re-expression. To examine the internalization pathway utilized by ligand-activated CXCR1, a CXCR1-GFP construct was transiently expressed in two different cell lines, HEK 293 and RBL-2H3 cells. While interleukin-8 stimulation promoted CXCR1 sequestration in RBL-2H3 cells, receptor internalization in HEK 293 cells required co-expression of G protein-coupled receptor kinase 2 and β-arrestin proteins. The importance of β-arrestins in CXCR1 internalization was confirmed by the ability of a dominant negative β-arrestin 1-V53D mutant to block internalization of CXCR1 in RBL-2H3 cells. A role for dynamin was also demonstrated by the lack of CXCR1 internalization in dynamin I-K44A dominant negative mutant-transfected RBL-2H3 cells. Agonist-promoted co-localization of transferrin and CXCR1-GFP in endosomes of RBL-2H3 cells confirmed that receptor internalization occurs via clathrin-coated vesicles. Our data provides a direct link between agonist-induced internalization of CXCR1 and a requirement for G protein-coupled receptor kinase 2, β-arrestins, and dynamin during this process. The functional role of neutrophils during acute inflammatory responses is regulated by two high affinity interleukin-8 receptors (CXCR1 and CXCR2) that are rapidly desensitized and internalized upon binding their cognate chemokine ligands. The efficient re-expression of CXCR1 on the surface of neutrophils following agonist-induced internalization suggests that CXCR1 surface receptor turnover may involve regulatory pathways and intracellular factors similar to those regulating β2-adrenergic receptor internalization and re-expression. To examine the internalization pathway utilized by ligand-activated CXCR1, a CXCR1-GFP construct was transiently expressed in two different cell lines, HEK 293 and RBL-2H3 cells. While interleukin-8 stimulation promoted CXCR1 sequestration in RBL-2H3 cells, receptor internalization in HEK 293 cells required co-expression of G protein-coupled receptor kinase 2 and β-arrestin proteins. The importance of β-arrestins in CXCR1 internalization was confirmed by the ability of a dominant negative β-arrestin 1-V53D mutant to block internalization of CXCR1 in RBL-2H3 cells. A role for dynamin was also demonstrated by the lack of CXCR1 internalization in dynamin I-K44A dominant negative mutant-transfected RBL-2H3 cells. Agonist-promoted co-localization of transferrin and CXCR1-GFP in endosomes of RBL-2H3 cells confirmed that receptor internalization occurs via clathrin-coated vesicles. Our data provides a direct link between agonist-induced internalization of CXCR1 and a requirement for G protein-coupled receptor kinase 2, β-arrestins, and dynamin during this process. A general characteristic of inflammatory responses is the migration of leukocytes from the blood to sites of injury or infection. A number of chemoattractants have been shown to cause the directed migration of leukocytes in vitro and in vivo. These include complement fragment C5a, formylated bacterial peptides (fMLP), arachidonic acid metabolites (LTB4), and a group of low-molecular weight pro-inflammatory cytokines known as chemokines (1Fernandez H.N. Henson P.M. Otani A. Hugli T.E. J. Immunol. 1978; 120: 109-115PubMed Google Scholar, 2Schiffmann E. Corcoran B.A. Wahl S.M. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 1059-1062Crossref PubMed Scopus (721) Google Scholar, 3Baggiolini M. Dewald B. Moser B. Adv. Immunol. 1994; 55: 97-179Crossref PubMed Scopus (2259) Google Scholar). The superfamily of chemokines is subdivided into different subsets based on the presence and positioning of highly conserved cysteine residues. The family, based on the configuration of the first two N-terminal located cysteines, is divided into CC, CXC, and CX3C subfamilies. All known neutrophil-targeted human chemokines belonging to the CXC subfamily (IL-8, 1The abbreviations used are: IL-8, interleukin-8; GPCR, guanine nucleotide-binding protein-coupled receptor; β2AR, β2-adrenergic receptor; GRK, G protein-coupled receptor kinases; GFP, green fluorescent protein; HEK, human embryonic kidney; RBL, rat basophilic leukemia; CCV, clathrin-coated vesicle; EMEM, Eagle's minimal essential medium; PBS, phosphate-buffered saline. 1The abbreviations used are: IL-8, interleukin-8; GPCR, guanine nucleotide-binding protein-coupled receptor; β2AR, β2-adrenergic receptor; GRK, G protein-coupled receptor kinases; GFP, green fluorescent protein; HEK, human embryonic kidney; RBL, rat basophilic leukemia; CCV, clathrin-coated vesicle; EMEM, Eagle's minimal essential medium; PBS, phosphate-buffered saline. GROα, GROβ, NAP-2, ENA78, and GCP-2) are defined by the presence of a glutamic acid-leucine-arginine motif (ELR motif) in the portion of the molecule that lies N-terminal to the first highly conserved cysteine, thus representing the ELR-CXC chemokine subclass of CXC chemokines (3Baggiolini M. Dewald B. Moser B. Adv. Immunol. 1994; 55: 97-179Crossref PubMed Scopus (2259) Google Scholar, 4Rollins B.J Blood. 1997; 90: 909-928Crossref PubMed Google Scholar, 5Murphy P.M. Cytokine Growth. Fact. Rev. 1996; 7: 47-64Crossref PubMed Scopus (281) Google Scholar). IL-8 and other neutrophil-directed chemokines stimulate neutrophils via specific seven-transmembrane guanine nucleotide-binding protein-coupled receptors (GPCRs) (5Murphy P.M. Cytokine Growth. Fact. Rev. 1996; 7: 47-64Crossref PubMed Scopus (281) Google Scholar, 7Kelvin D.J. Michiel D.F. Johnston J.A. Lloyd A.R. Sprenger H. Oppenheim J.J. Wang J.M. J. Leukocyte Biol. 1993; 54: 604-612Crossref PubMed Scopus (145) Google Scholar). The two human IL-8 receptors, CXCR1 and CXCR2, have 77% overall sequence homology. The two receptor subtypes differ notably in their N-terminal extracellular domains, as well as in their C-terminal intracellular domains, and possess differences in their ligand specificities. CXCR1 displays greater ligand specificity by binding to IL-8 and GCP-2 with high affinity, whereas CXCR2 binds with high affinity multiple CXC chemokines in addition to IL-8, including ENA 78, NAP-2, GROα, and GROβ (6Murphy P.M. Semin. Hematol. 1997; 34: 311-318PubMed Google Scholar, 7Kelvin D.J. Michiel D.F. Johnston J.A. Lloyd A.R. Sprenger H. Oppenheim J.J. Wang J.M. J. Leukocyte Biol. 1993; 54: 604-612Crossref PubMed Scopus (145) Google Scholar, 8Holmes W.E. Lee J. Kuang W.J. Rice G.C. Wood W.I. Science. 1992; 253: 1278-1280Crossref Scopus (907) Google Scholar, 9Lee J. Horuk R. Rice G.C. Bennett G.L. Camerato T. Wood W.I. J. Biol. Chem. 1992; 267: 16283-16287Abstract Full Text PDF PubMed Google Scholar). Binding of the ligand to high affinity IL-8 receptors initiates a variety of cellular responses, including calcium translocation, chemotaxis, alterations in cytoskeletal architecture as well as cellular morphology, degranulation, and respiratory burst activation (3Baggiolini M. Dewald B. Moser B. Adv. Immunol. 1994; 55: 97-179Crossref PubMed Scopus (2259) Google Scholar, 10Hammond M. Lapointe G. Feucht P.S. Hilt S. Gallegos C. Gardon C. J. Immunol. 1995; 155: 1428-1433PubMed Google Scholar, 11Sham R.L. Phatak P.D. Ihne T.P. Abboud C.N. Packman C.H. Blood. 1993; 82: 2546-2551Crossref PubMed Google Scholar, 12Camps M. Carrozi A. Schnabel P. Scheer A. Parker P.J. Gierschik P. Nature. 1992; 360: 684-686Crossref PubMed Scopus (513) Google Scholar). ELR-CXC chemokines are produced by a variety of cell types including monocytes, T lymphocytes, fibroblasts, and endothelial cells (3Baggiolini M. Dewald B. Moser B. Adv. Immunol. 1994; 55: 97-179Crossref PubMed Scopus (2259) Google Scholar, 5Murphy P.M. Cytokine Growth. Fact. Rev. 1996; 7: 47-64Crossref PubMed Scopus (281) Google Scholar, 6Murphy P.M. Semin. Hematol. 1997; 34: 311-318PubMed Google Scholar). It has been well documented that IL-8 receptors become rapidly desensitized and internalized upon agonist stimulation (13Samanta A.K. Oppenheim J.J. Matsushima K. J. Biol. Chem. 1990; 265: 183-189Abstract Full Text PDF PubMed Google Scholar, 14Chuntharapai A. Kim K.J. J. Immunol. 1995; 155: 2587-2594PubMed Google Scholar). The molecular mechanism(s) and cellular factors required for translocation of these agonist-occupied receptors from the membrane to cytosolic compartments are not well characterized. However, the rapid sequestration and re-expression of CXCR1 (14Chuntharapai A. Kim K.J. J. Immunol. 1995; 155: 2587-2594PubMed Google Scholar, 16Sabroe I. Williams T.J. Hebert C.A. Collins P.D. J. Immunol. 1997; 158: 1361-1369PubMed Google Scholar) is similar to the well described model of β2-adrenergic receptor (β2-AR) regulation. In the case of β2-AR, agonist binding induces a change in the receptor conformation, which is necessary for the interaction of the receptor with G protein-coupled receptor kinases (GRKs) (17Premont T.P. Inglese J. Lefkowitz R.J. FASEB J. 1995; 9: 175-182Crossref PubMed Scopus (469) Google Scholar, 19Ferguson S.S.G. Barak L.S. Zhang J. Caron M.G. Can. J. Physiol. Pharmacol. 1996; 74: 1095-1110Crossref PubMed Scopus (318) Google Scholar). GRK-mediated phosphorylation of the β2-AR C terminus promotes binding of arrestin proteins (β-arrestins) which when bound, elicit uncoupling of the receptor from its G protein (18Lohse M.J. Benovic J.L. Codina J. Caron M.G. Lefkowitz R.J. Science. 1990; 248: 1547-1550Crossref PubMed Scopus (900) Google Scholar, 19Ferguson S.S.G. Barak L.S. Zhang J. Caron M.G. Can. J. Physiol. Pharmacol. 1996; 74: 1095-1110Crossref PubMed Scopus (318) Google Scholar, 20Ferguson S.S.G. Downey III, W.E. Colapietro A.M. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-366Crossref PubMed Scopus (841) Google Scholar). Recent data suggests that the synergistic action of cellular GRKs and β-arrestins determines the kinetics of β2-AR internalization (21Menard L. Ferguson S.S.G. Zhang J. Lin F.T. Lefkowitz R.J. Caron M.G. Barak L.S. Mol. Pharmacol. 1997; 51: 800-808Crossref PubMed Scopus (213) Google Scholar). Moreover, it was demonstrated that β-arrestins serve as adaptor proteins, specifically targeting agonist-occupied receptors to clathrin-coated vesicles (CCVs) (19Ferguson S.S.G. Barak L.S. Zhang J. Caron M.G. Can. J. Physiol. Pharmacol. 1996; 74: 1095-1110Crossref PubMed Scopus (318) Google Scholar, 20Ferguson S.S.G. Downey III, W.E. Colapietro A.M. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-366Crossref PubMed Scopus (841) Google Scholar, 22Goodman O.B. Krupnick J.G. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Crossref PubMed Scopus (1156) Google Scholar). A critical step in receptor-mediated endocytosis of β2-AR is the translocation of CCVs saturated with agonist-occupied receptor to the cytosol, which is a dynamin-regulated event (23Zhang J. Ferguson S.S.G. Barak L.S. Menard L. Caron M.G. J. Biol. Chem. 1996; 271: 18302-18305Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). Desensitized β2-ARs, internalized via CCVs, are thought to be resensitized in the acidified endosomal environment and recycled back to the cell surface to re-establish normal receptor signaling (24Zhang J. Barak L.S. Winkler K.E. Caron M.G. Ferguson S.S.G. J. Biol. Chem. 1997; 272: 27005-27014Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). In the present work we examined the role of GRK2, β-arrestins, and dynamin in regulating CXCR1 internalization. For this purpose a CXCR1-green fluorescent protein (GFP) construct (CXCR1-GFP) was transiently expressed in human embryonic kidney 293 (HEK 293) and rat basophilic leukemia 2H3 (RBL-2H3) cell lines. We demonstrate that GRK2, β-arrestins, and dynamin are required for rapid agonist-induced internalization of CXCR1. HEK 293 and RBL-2H3 cell lines were obtained from American Type Culture Collection (Manassas, VA). Dulbecco's modified essential medium and Eagle's modified essential medium (EMEM) were purchased from Biowhittaker (Walkerswille, MD). Chemiluminescent substrates and horseradish peroxidase-coupled donkey anti-rabbit antibody were purchased from Amersham (Amersham International). The plasmid containing a variant of a green fluorescence protein (pEGFP-N1) and a green fluorescence protein directed polyclonal antibody were purchased from CLONTECH (Palo Alto, CA). Human recombinant interleukin-8 (IL-8) was obtained from R&D Systems (Minneapolis, MN) and 125I-IL-8 was obtained from Amersham. Texas Red-transferrin conjugates were purchased from Molecular Probes (Eugene, OR). RNA from human neutrophils was Molecular and CXCR1 to the The sequence of CXCR1 was and were and with and into The construct was the that was by to sequence HEK 293 cells were in Dulbecco's modified essential medium whereas RBL-2H3 cells were in EMEM, containing and of in a of and HEK 293 cells were to in a of 2 and RBL-2H3 cells were a of cells to HEK 293 cells were transiently with of CXCR1-GFP and of rat β-arrestin rat β-arrestin 2, of rat For RBL-2H3 cells, to the cells were and in and transiently with of CXCR1-GFP or with of rat β-arrestin 1-V53D or of rat dynamin lines were with following the the cells were in medium for to For RBL-2H3 cells were to in and with of CXCR1-GFP in of cells were and in with of The was and were was confirmed by of cells RBL-2H3 cells were in in were with containing and and in the RBL-2H3 and RBL-2H3 cells CXCR1-GFP were with and for The was with of with the cells were with the and in the and in on for the of the well was and for of the were and the in was a of was in of RBL-2H3 cells. were as described for the and in for and RBL-2H3 cells CXCR1-GFP were with IL-8 for The was by on for The of the was by of the medium with of in for the of the of a was and the was was on a a with the of HEK 293 and RBL-2H3 cells were in For the cells were with of IL-8 and and following agonist stimulation were in to To the of protein that occurs during the of RBL-2H3 cells transiently CXCR1-GFP were first with for and with following agonist were For RBL-2H3 cells transiently CXCR1-GFP were with IL-8 and with Texas Red-transferrin conjugates for The was by the cells with The cells were in and was as described HEK 293 cells transiently and GRK2, and GRK2, and β-arrestin were with IL-8 for The cells were with from by and for The cell was in of A 2 on for and a were by for The was on a and in and for The was and the containing endosomes was in and for The were in of A containing 2 and of protein was in the cell of HEK 293 and RBL-2H3 cells were protein with as a of β-arrestin β-arrestin 2, and 2 in HEK 293 and RBL-2H3 cells were examined specific polyclonal as described S.S.G. Downey III, W.E. Colapietro A.M. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-366Crossref PubMed Scopus (841) Google Scholar). of cell protein were on a and membrane The of β-arrestin β-arrestin 2, and 2 were 2 and polyclonal a of and horseradish anti-rabbit the to The of β-arrestin β-arrestin 2, and 2 in RBL-2H3 cells were to their in HEK 293 cells. of CXCR1-GFP in the were polyclonal antibody The membrane and was was and are the from with CXCR1-GFP were for protein as by membrane fluorescence in of the RBL-2H3 and of the HEK 293 cells by In cells the CXCR1-GFP in the that internalization of CXCR1-GFP in a of a agonist-occupied CXCR1-GFP conjugates in specific from the membrane to the data in RBL-2H3 cells a of in membrane and a in fluorescence in to agonist stimulation the was a of membrane in to IL-8 in RBL-2H3 cells transiently the CXCR1-GFP construct not of the expressed receptor was internalized of RBL-2H3 cells transiently the CXCR1-GFP construct with in rapid CXCR1-GFP sequestration with membrane fluorescence that membrane fluorescence was to of CXCR1-GFP conjugates not cells of CXCR1-GFP the CXCR1-GFP cells internalized 125I-IL-8 whereas CXCR1-GFP cells to 125I-IL-8 that internalization of CXCR1-GFP is agonist and which is similar to IL-8 receptor internalization in neutrophils (14Chuntharapai A. Kim K.J. J. Immunol. 1995; 155: 2587-2594PubMed Google Scholar). To CXCR1-GFP functional responses, we on IL-8 and CXCR1-GFP cells. IL-8 stimulation of CXCR1-GFP cells in a of with a from RBL-2H3 cells with IL-8 with a chemokine that not CXCR1, not These that CXCR1-GFP expressed in RBL-2H3 cells of the receptor expressed in the receptor that in agonist-induced and In to RBL-2H3 cells, HEK 293 cells transiently the protein construct not CXCR1-GFP when with IL-8 2 I. Zhang J. Ferguson S.S.G. P.D. Caron M.G. J. 1997; PubMed Scopus Google Scholar) have demonstrated that HEK 293 cells of β-arrestins and GRKs for internalization of we co-expression of these two of with CXCR1-GFP agonist-induced receptor internalization in HEK 293 cells. of CXCR1-GFP with 2 or β-arrestin 2 in HEK 293 cells to IL-8 CXCR1-GFP internalization. However, of CXCR1-GFP with and β-arrestins in receptor internalization 2 a of cytosolic fluorescence and a in membrane fluorescence 2 The cytosolic receptor fluorescence was with receptor of the intracellular vesicles. were obtained with β-arrestin 2, of the β-arrestin family, with and CXCR1-GFP 2 These that GRKs and β-arrestins are required for CXCR1 internalization. of GRK2, β-arrestin and β-arrestin 2 from HEK 293 cells and RBL-2H3 cells A and a in β-arrestin β-arrestin 2, and between HEK 293 cells and RBL-2H3 cells. The of and β-arrestin 2 in RBL-2H3 cells CXCR1-GFP agonist-induced internalization in RBL-2H3 cells the requirement for co-expression with GRKs or To the role of β-arrestins in CXCR1-GFP internalization in RBL-2H3 cells we CXCR1-GFP with the β-arrestin 1-V53D dominant negative mutant in RBL-2H3 cells and with In the presence of β-arrestin 1-V53D was of membrane fluorescence that IL-8 stimulation and was in to cells CXCR1-GFP or cells CXCR1-GFP and β-arrestin not which receptor internalization following IL-8 These complement the obtained with HEK 293 cells and demonstrate a role of β-arrestins in agonist-induced CXCR1 internalization. are thought to as proteins in to CCVs O.B. Krupnick J.G. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Crossref PubMed Scopus (1156) Google Scholar, J. Barak L.S. Winkler K.E. Caron M.G. Ferguson S.S.G. J. Biol. Chem. 1997; 272: 27005-27014Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, L.S. Ferguson S.S.G. Zhang J. Caron M.G. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, L. Gagnon A.W. Penn R.B. Benovic J.L. J. Biol. Chem. 1997; Full Text Full Text PDF Scopus Google Scholar). stimulation promotes the of CXCR1-GFP containing which are from the membrane and into compartments (19Ferguson S.S.G. Barak L.S. Zhang J. Caron M.G. Can. J. Physiol. Pharmacol. 1996; 74: 1095-1110Crossref PubMed Scopus (318) Google Scholar, J. Barak L.S. Winkler K.E. Caron M.G. Ferguson S.S.G. J. Biol. Chem. 1997; 272: 27005-27014Abstract Full Text Full Text PDF PubMed Scopus (219) Google L. Gagnon A.W. Penn R.B. Benovic J.L. J. Biol. Chem. 1997; Full Text Full Text PDF Scopus Google Scholar). The or of the vesicles from the membrane is upon a containing molecule H. 1996; PubMed Scopus Google Scholar, J. 1997; PubMed Scopus Google Scholar, H. T. J. Biol. 1994; PubMed Scopus Google Scholar). The dominant negative dynamin I-K44A mutant has been utilized in are internalized via a pathway We CXCR1 required dynamin for agonist-induced receptor internalization by CXCR1-GFP with the dynamin I-K44A dominant negative The of dynamin I-K44A of CXCR1-GFP from the membrane to the in cells the dynamin I-K44A mutant not from the surface of the membrane These that agonist-induced internalization of CXCR1 occurs via CCVs and functional β-arrestins and dynamin To and the of vesicles that agonist-occupied CXCR1-GFP from the membrane to we RBL-2H3 cells transiently the construct with a Texas Red-transferrin has been shown to receptor-mediated endocytosis CCVs upon binding to its cognate transferrin receptor and it has been described as a endosomal J. PubMed Scopus Google Scholar, P. C. Semin. Hematol. Google Scholar). stimulation promoted of CXCR1-GFP and transferrin CCVs whereas RBL-2H3 cells transiently CXCR1-GFP not These were by of the from transiently HEK 293 cells CXCR1-GFP and GRK2, and β-arrestin that were or with A in CXCR1-GFP was in the from IL-8 HEK 293 cells GRK2, and β-arrestin However, a in CXCR1-GFP was in the from HEK 293 cells in the of and β-arrestin that and β-arrestin the of CXCR1 internalization. these a model for CXCR1 sequestration via clathrin-coated that are regulated by β-arrestins, and of high CXCR1-GFP in the endosomal from HEK 293 cells transiently GRK2, and β-arrestin of CXCR1-GFP in from HEK 293 cells transiently CXCR1-GFP and and GRK2, and β-arrestin 2 and were polyclonal of chemokine receptor and sequestration is for regulating to chemokine have demonstrated that of neutrophils to high of ELR-CXC chemokines the neutrophils to chemokine The of neutrophils following stimulation with high chemokine to be upon and sequestration of CXCR1, CXCR2, or IL-8 receptor IL-8 that sites were rapidly from the surface following stimulation of high of IL-8 (13Samanta A.K. Oppenheim J.J. Matsushima K. J. Biol. Chem. 1990; 265: 183-189Abstract Full Text PDF PubMed Google Scholar). These were confirmed by work directed the of CXCR1 and CXCR2, that demonstrated a rapid of CXCR1 and CXCR2 following stimulation of neutrophils with high of ELR-CXC chemokines (14Chuntharapai A. Kim K.J. J. Immunol. 1995; 155: 2587-2594PubMed Google Scholar, L. R. G. M. J.G. D.J. J. Immunol. Google Scholar). Recent by H. B. R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) have demonstrated that phosphorylation of critical in the C-terminal of CXCR1 is for internalization of the receptor following agonist Our work these by that is necessary for internalization of CXCR1 in HEK 293 cells 2 and is a kinase and a of a and internalization by located the of receptors (17Premont T.P. Inglese J. Lefkowitz R.J. FASEB J. 1995; 9: 175-182Crossref PubMed Scopus (469) Google Scholar). is expressed in human leukocytes M. G. G. A. J. Biol. Chem. 267: and data not and may the kinase for CXCR1 phosphorylation in of the other of the and may serve the in regulating phosphorylation of CXCR1 in a of the chemokine of receptors B.J Blood. 1997; 90: 909-928Crossref PubMed Google is by and that phosphorylation a of and CXC chemokine receptor I. Zhang J. Ferguson S.S.G. P.D. Caron M.G. J. 1997; PubMed Scopus Google Scholar). While phosphorylation a critical step in regulating the and internalization of a of it is the β-arrestin proteins which the translocation of from the membrane to Our data in the present β-arrestins as of CXCR1 internalization in to agonist has been two cells lines two different HEK 293 cells which have of β-arrestins of β-arrestin or β-arrestin 2 for agonist internalization. In to HEK 293 cells, RBL-2H3 cells of β-arrestins and not of β-arrestins for CXCR1 internalization However, agonist-induced internalization of CXCR1 be by the dominant negative β-arrestin 1-V53D mutant in RBL-2H3 cells and These for β-arrestin of agonist-induced CXCR1 internalization. it is also from that cellular factors other and β-arrestin are in the CXCR1 internalization have been described as proteins in the or of CCVs from the membrane by which H. 1996; PubMed Scopus Google Scholar, J. 1997; PubMed Scopus Google Scholar). In to the which is to endocytosis (23Zhang J. Ferguson S.S.G. Barak L.S. Menard L. Caron M.G. J. Biol. Chem. 1996; 271: 18302-18305Abstract Full Text Full Text PDF PubMed Scopus (398) Google that in the presence of dynamin I-K44A CCVs saturated with agonist-occupied receptor are not from the membrane into the cytosolic CXCR1 to internalization and sequestration a and internalization pathway similar to other S.S.G. Downey III, W.E. Colapietro A.M. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-366Crossref PubMed Scopus (841) Google Scholar, L. Gagnon A.W. Penn R.B. Benovic J.L. J. Biol. Chem. 1997; Full Text Full Text PDF Scopus Google Scholar, I. Zhang J. Ferguson S.S.G. P.D. Caron M.G. J. 1997; PubMed Scopus Google Scholar). is by two of of transferrin and CXCR1-GFP in endosomal vesicles of RBL-2H3 cells and of CXCR1-GFP to the following IL-8 stimulation in HEK 293 cells transiently GRK2, and β-arrestin CXCR1 and CXCR2 77% acid and elicit similar functional responses to have pathways for internalization and (14Chuntharapai A. Kim K.J. J. Immunol. 1995; 155: 2587-2594PubMed Google Scholar) that CXCR1 not CXCR2 was recycled back to the membrane following agonist-induced internalization. The for the of CXCR1 and CXCR2 is differences in the C-terminal may a role in the two proteins intracellular H. B. J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). In this CXCR1 and CXCR2 may have for β-arrestin proteins, which CXCR1 and CXCR2 to different intracellular data in suggests that CXCR2 internalization is regulated from CXCR1 in cells. The role of β-arrestins in regulating CXCR1 and CXCR2 It is to that β-arrestins as signaling have a role for β-arrestins in the activation of kinases Ferguson S.S.G. W.E. Lin K. Caron M.G. Lefkowitz R.J. Science. PubMed Scopus Google Scholar). it is that of the functional responses by CXCR1 are to by β-arrestins to the CXCR1 While are for internalization including the and and to and pathways (23Zhang J. Ferguson S.S.G. Barak L.S. Menard L. Caron M.G. J. Biol. Chem. 1996; 271: 18302-18305Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar, P. J. Biol. PubMed Scopus Google Scholar, J. Biol. PubMed Scopus Google Scholar). We present a model for CXCR1 chemokine receptor internalization we have demonstrated that GRK2, and dynamin are necessary for the of CXCR1 into the IL-8 C-terminal on CXCR1 β-arrestins to receptor to containing data that dynamin is required to CCVs containing CXCR1 and of the receptor into the The importance of chemokine receptor internalization may be to serve as a of the of leukocytes of high to inflammatory their migration and from the of These into the factors in chemokine receptor into the cell and thus may of the inflammatory process. We M. for critical of this and for