Key points are not available for this paper at this time.
We have characterized the ligand-enhanced phosphorylation of the CXC chemokine receptor-2 (CXCR2) in a series of clonal 3ASubE cell lines expressing receptors truncated or mutated in the carboxyl-terminal domain. Truncation of CXCR2 by substitution of a stop codon for Ser-342 (342T) or Ser-331 (331T) results in total loss of melanoma growth stimulatory activity/growth-related protein (MGSA/GRO)-enhanced receptor phosphorylation, which cannot be explained based upon altered ligand binding affinity or receptor number. 3ASubE cells expressing 342T or CXCR2 with mutation of Ser-342, −346, −347, and −348 to alanine (4A) exhibit strong mobilization of Ca2+ in response to ligand (interleukin-8 or MGSA/GRO), with a recovery phase significantly slower than that of cells expressing wild type (WT) CXCR2. In contrast to the WT CXCR2, which is 93% desensitized by 20 nM ligand, the 331T, 342T, and 4A CXCR2 mutants do not undergo significant ligand-induced desensitization, and respond to a second ligand challenge by mobilizing Ca2+. The 3ASubE cells expressing CXCR2 with mutation of Ser-346, −347, and −348 to alanine, or with mutation of only one serine in this domain, continue to be phosphorylated in response to ligand and are 60–70% desensitized following the initial ligand challenge. WT CXCR2 phosphorylation and desensitization occur in 30 min. The 342T CXCR2 receptor is not sequestered in response to ligand. These data demonstrate that for CXCR2, specific residues along the carboxyl-terminal domain of the receptor mediate receptor desensitization and sequestration in response to ligand.DISCUSSIONThere is now considerable evidence that ligand binding to transmembrane-spanning receptors stimulates receptor phosphorylation on the carboxyl-terminal domain of the receptor and that this event coincides with receptor desensitization, sequestration, and in some instances degradation or recycling (3Chuntharapai A. Kim K.J. J. Immunol. 1995; 155: 2587-2594PubMed Google Scholar, 4Mueller S.G. Schraw W.P. Richmond A. J. Biol. Chem. 1994; 269: 1973-1980Abstract Full Text PDF PubMed Google Scholar, 5Mueller S.G. Schraw W.P. Richmond A. J. Biol. Chem. 1995; 270: 10439-10448Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 6Pitcher J. Lohse M.J. Codina J. Caron M.G. Lefkowitz R.J. Biochemistry. 1992; 31: 3193-3197Crossref PubMed Scopus (149) Google Scholar, 7Pei G. Samama P. Lohse M. Wang M. Codina J. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2699-2702Crossref PubMed Scopus (138) Google Scholar, 8Ferguson S.S.G. Menard L. Barak L.S. Koch W.J. Colapietro A.-M. Caron M.G. J. Biol. Chem. 1995; 270: 24782-24789Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Both CC and CXC chemokine receptors are phosphorylated in response to ligand binding, and multiple serine or serine and threonine residues are phosphorylated (4Mueller S.G. Schraw W.P. Richmond A. J. Biol. Chem. 1994; 269: 1973-1980Abstract Full Text PDF PubMed Google Scholar, 10Franci C. Gosling J. Tsou C-L. Coughlin S.R. Charo I.F. J. Immunol. 1996; 157: 5606-5612PubMed Google Scholar, 11Richardson R.M. DuBose R.A. Ali H. Tomhave E.D. Haribabu D. Snyderman R. Biochemistry. 1995; 34: 14193-14201Crossref PubMed Scopus (88) Google Scholar). Richardson et al. (11Richardson R.M. DuBose R.A. Ali H. Tomhave E.D. Haribabu D. Snyderman R. Biochemistry. 1995; 34: 14193-14201Crossref PubMed Scopus (88) Google Scholar) have shown that the phosphorylation of CXCR1 is accompanied by receptor desensitization. CXCR2 expressed in CHO cells is reported to be internalized more rapidly than CXCR1, and MGSA induces this internalization more slowly than does IL-8 (12Prado G.N. Suzuki H. Wilkinson N. Cousins B. Navarro J. J. Biol. Chem. 1996; 271: 19186-19190Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Moreover, truncation of CXCR1 eliminates ~60% of the ligand-induced internalization of the receptor (12Prado G.N. Suzuki H. Wilkinson N. Cousins B. Navarro J. J. Biol. Chem. 1996; 271: 19186-19190Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). In contrast, neutrophil CXCR1 and CXCR2 are both rapidly down-modulated by ligand (5 min), although considerably more ligand is required to down-modulate CXCR1 than CXCR2 (3Chuntharapai A. Kim K.J. J. Immunol. 1995; 155: 2587-2594PubMed Google Scholar). CXCR2 recycles more slowly than the CXCR1; moreover, only about 40% of the CXCR2 recycles during a 3-h culture period (3Chuntharapai A. Kim K.J. J. Immunol. 1995; 155: 2587-2594PubMed Google Scholar), while CXCR1 fully recovers 1.5 h after treatment with the IL-8 ligand in neutrophils. Our studies with 3ASubE cells show that desensitization and phosphorylation of WT CXCR2 occurs within 30–0 s after ligand treatment, while sequestration occurs much more slowly, requiring 30–0 min. Thus, CXCR2 clearance in 3ASubE cells is different from that reported for CHO cells (12Prado G.N. Suzuki H. Wilkinson N. Cousins B. Navarro J. J. Biol. Chem. 1996; 271: 19186-19190Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). We have also observed that after 6 h of treatment with 25 nM MGSA/GRO, 3ASubE cells expressing WT CXCR2 exhibit only ~50% reduction in the total receptor detected by Western blot, suggesting that either only a percentage of the receptors that bind ligand undergo degradation after sequestration or, alternatively, newly translated CXCR2 rapidly replenishes the receptor pool. A recent study of the N-formyl peptide receptor demonstrates a similar dynamic state of receptor sequestration, counterbalanced by the reappearance of receptors at the membrane (13Hoffman J.F. Linderman J.J. Omann G.M. J. Biol. Chem. 1996; 271: 18394-18404Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar).3ASubE cells expressing wild type and mutant CXCR2 exhibit only subtle differences in the time course for desensitization or receptor sequestration produced by IL-8 and MGSA/GRO. However, larger differences in IL-8- versus MGSA/GRO-induced calcium signaling in 293 cells expressing transfected CXCR2 were recently observed by Damaj et al. (21Damaj B.B. McColl S.R. Neote K. Hebert C.A. Naccache P.H. J. Biol. Chem. 1996; 271: 20540-20544Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), who suggested that MGSA/GRO elicits a stronger influx of Ca2+ than does IL-8. Differences in IL-8 versus MGSA/GRO induction of sequestration of CXCR2 were observed by Prado et al. in transfected CHO cells (11Richardson R.M. DuBose R.A. Ali H. Tomhave E.D. Haribabu D. Snyderman R. Biochemistry. 1995; 34: 14193-14201Crossref PubMed Scopus (88) Google Scholar). The basis for the different results between these two studies and our study is unclear but could reflect differences in the specific activity or potency of the ligand preparations or differences in the manner in which the various cell types process the receptors. More in keeping with our own findings, Ahuja et al. (22Ahuja S.K. Lee J.C. Murphy P.M. J. Biol. Chem. 1996; 271: 225-232Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 23Ahuja S.K. Murphy P.M. J. Biol. Chem. 1996; 271: 20545-20550Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar) reported similar profiles of calcium mobilization for IL-8 and MGSA/GRO proteins in stably transfected 293 cells expressing CXCR2, although MGSA/GRO was unable to totally desensitize receptors to a second weaker response to IL-8.The β-adrenergic receptors are phosphorylated along the carboxyl tail by β-adrenergic receptor kinase and then assume a conformation that allows association with an arrestin (14Ferguson 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 (839) Google Scholar). This association with arrestin is thought to be necessary for both desensitization and for sequestration. In the case of the β2-adrenergic receptor, tyrosine phosphorylation of Tyr-326 is involved in facilitation of sequestration of this receptor (14Ferguson 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 (839) Google Scholar). We observe no tyrosine phosphorylation of CXCR2 in response to ligand or phorbol-ester stimulation. While some investigators have reported that sequestration is not dependent upon phosphorylation, others have shown that overexpression of β-adrenergic receptor kinase-1 can facilitate the sequestration of the M2-muscarinic receptor. Moreover, for the M2 receptor, desensitization has been shown to proceed in the absence of sequestration (15Yu S.S. Lefkowitz R.J. Hausdorff W.P. J. Biol. Chem. 1993; 268: 337-341Abstract Full Text PDF PubMed Google Scholar, 16Barak L.S. Tiberi M. Freedman N.J. Kwatra M.M. Lefkowitz R.J. Caron M.G. J. Biol. Chem. 1994; 269: 2790-2795Abstract Full Text PDF PubMed Google Scholar). A number of seven-transmembrane G protein-coupled receptors show loss of both phosphorylation and sequestration when the carboxyl-terminal domain is deleted (17Lattion A.-L. Diviani D. Cotecchia S. J. Biol. Chem. 1994; 269: 22887-22893Abstract Full Text PDF PubMed Google Scholar, 18Huang Z. Chen Y. Nissenson R.A. J. Biol. Chem. 1995; 270: 151-156Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 19Thomas W.G. Thekkumkara T.J. Motel T.J. Baker K.M. J. Biol. Chem. 1995; 270: 207-213Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Our studies on CXCR2 phosphorylation, desensitization, and sequestration demonstrate that the loss of four specific serine residues involved in ligand-stimulated receptor phosphorylation results in markedly receptor desensitization and loss of receptor sequestration but no loss of ligand-induced However, we are unable to to one specific phosphorylation a for desensitization or sequestration. appear to be A similar of serine and threonine residues CXCR1 desensitization in the cell (11Richardson R.M. DuBose R.A. Ali H. Tomhave E.D. Haribabu D. Snyderman R. Biochemistry. 1995; 34: 14193-14201Crossref PubMed Scopus (88) Google Scholar). However, is to that desensitization does not receptor Richardson et al. R.M. Ali H. Tomhave E.D. Haribabu B. Snyderman R. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar) have recently shown that of an receptor can and both the receptor and and IL-8 also the receptor based upon Ca2+ mobilization and but this is not accompanied by phosphorylation of the receptor. Richardson et al. R.M. Ali H. Tomhave E.D. Haribabu B. Snyderman R. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar) that these data that of of receptor G protein be involved in in the absence of receptor phosphorylation R.M. Ali H. Tomhave E.D. Haribabu B. Snyderman R. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google have observed only serine phosphorylation of CXCR2 (4Mueller S.G. Schraw W.P. Richmond A. J. Biol. Chem. 1994; 269: 1973-1980Abstract Full Text PDF PubMed Google Scholar, 5Mueller S.G. Schraw W.P. Richmond A. J. Biol. Chem. 1995; 270: 10439-10448Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Moreover, acid of the mutant of CXCR2 revealed only serine phosphorylation not Truncation and serine mutants of CXCR2 that a loss of ligand-induced CXCR2 phosphorylation also a loss of CXCR2 Thus, in 3ASubE cells CXCR2 is from CXCR1 in cells, threonine phosphorylation also be and phorbol-ester of receptor phosphorylation to from that by ligand (11Richardson R.M. DuBose R.A. Ali H. Tomhave E.D. Haribabu D. Snyderman R. Biochemistry. 1995; 34: 14193-14201Crossref PubMed Scopus (88) Google Scholar). the CXCR1, phosphorylation of CXCR2 along the carboxyl-terminal domain to be in the desensitization and loss of amino acid residues involved in this phosphorylation results in of the receptor desensitization as as receptor sequestration in response to ligand. Moreover, these two receptors appear to exhibit differences in the time course of sequestration and recycling (3Chuntharapai A. Kim K.J. J. Immunol. 1995; 155: 2587-2594PubMed Google Scholar). These are to be in the of the an response to is required to on and in an ligand for some INTRODUCTIONThe CXC chemokine, 1The abbreviations used are: CXCchemokine, chemokine with the first two amino acids separated by an intervening amino acid3ACXCR2 mutant with serine to alanine substitutions at positions 346, 347, and 3484ACXCR2 mutant with serine to alanine substitutions at positions 342, 346, 347, and 348342TCXCR2 truncated at the Ser-342 by placing a stop codon at Ser-342331TCXCR2 truncated at Ser-331 by placement of a stop codon in place of Ser352TCXCR2 truncated at Ser-352 by placing a stop codon at Ser-352293 cellshuman embryonic kidney cell line293T2human embryonic kidney 293 cells expressing transfected human CXCR2CCchemokines, chemokines with the first two cysteines positioned side-by-sideCMVcytomegalovirusCXCR1receptor for CXC chemokines formerly referred to as IL-8 receptor ACXCR2receptor for CXC chemokines formerly defined as IL-8 receptor BC5acomplement fragment 5aDMEMDulbecco's modified Eagle's mediumFBSfetal bovine serumfMLPN-formyl-methionyl-leucyl-phenylalanineFURA-2fluorescence indicator for free calciumIL-8interleukin-8MGSA/GROmelanoma growth-stimulatory activity/growth-related proteinPBSphosphate-buffered salineTPA12-O-tetradecanoylphorbol-13-acetateWTwild type. IL-8, specifically binds the CXC chemokine receptor, CXCR1, while a second receptor, CXCR2, is shared by IL-8, MGSA/GRO, and several other CXC chemokines (1Richmond A. Shattuck R.L. Horuk R. Chemoattractant Ligands and Their Receptors.CRC Press, Inc. 1996; : 87-124Google Scholar). In human neutrophils, a 10-min treatment with IL-8 induces the internalization of greater than 90% of the CXC chemokine receptors, CXCR1 and CXCR2 (2Samanta A.K. Oppenheim J.J. Matsushima K. J. Biol. Chem. 1990; 265: 183-189Abstract Full Text PDF PubMed Google Scholar). Low MGSA/GRO concentrations (0.2 nM) down-regulated 50% of CXCR2 expressed on neutrophils, while 7–13-fold higher IL-8 concentrations are required to down-regulate 50% of CXCR1 (3Chuntharapai A. Kim K.J. J. Immunol. 1995; 155: 2587-2594PubMed Google Scholar). Cell surface expression of CXCR1 is 100% restored after 1.5 h, while only 40% of the initial CXCR2 expression can be detected after 3 h of incubation following ligand treatment (3Chuntharapai A. Kim K.J. J. Immunol. 1995; 155: 2587-2594PubMed Google Scholar). We have previously demonstrated in both 3ASubE and 293 stable transfectants expressing CXCR2 that ligand binding results in the phosphorylation of CXCR2 on serine residues (4Mueller S.G. Schraw W.P. Richmond A. J. Biol. Chem. 1994; 269: 1973-1980Abstract Full Text PDF PubMed Google Scholar, 5Mueller S.G. Schraw W.P. Richmond A. J. Biol. Chem. 1995; 270: 10439-10448Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Within 1 min after ligand binding, MGSA/GRO stimulates the phosphorylation of CXCR2, and after prolonged treatment with MGSA/GRO ~40% of the receptors are degraded (4Mueller S.G. Schraw W.P. Richmond A. J. Biol. Chem. 1994; 269: 1973-1980Abstract Full Text PDF PubMed Google Scholar). Phorbol ester treatment (100 nM for 2 h) of 3ASubE cells expressing CXCR2 also results in serine phosphorylation, down-regulation, and degradation of the receptor (5Mueller S.G. Schraw W.P. Richmond A. J. Biol. Chem. 1995; 270: 10439-10448Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). These data suggest that phosphorylation of CXCR2 induces receptor internalization (sequestration) and degradation, as has been observed in certain adrenergic receptors (6Pitcher J. Lohse M.J. Codina J. Caron M.G. Lefkowitz R.J. Biochemistry. 1992; 31: 3193-3197Crossref PubMed Scopus (149) Google Scholar, 7Pei G. Samama P. Lohse M. Wang M. Codina J. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2699-2702Crossref PubMed Scopus (138) Google Scholar, 8Ferguson S.S.G. Menard L. Barak L.S. Koch W.J. Colapietro A.-M. Caron M.G. J. Biol. Chem. 1995; 270: 24782-24789Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Recently, Ben-Baruch et al. (9Ben-Baruch A. Xu L. Young P.R. Bengali K. Oppenheim J.J. Wang J.M. J. Biol. Chem. 1995; 270: 22123-22128Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) demonstrated that 293 cells expressing CXCR2 exhibiting a deletion of amino acids 317–355 from the carboxyl-terminal domain do not exhibit chemotaxis in response to IL-8 (9Ben-Baruch A. Xu L. Young P.R. Bengali K. Oppenheim J.J. Wang J.M. J. Biol. Chem. 1995; 270: 22123-22128Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Since ligand did induce migration of 293 cells expressing CXCR2 with a deletion of residues 325–355, residues 317–324 appear to be essential for signal transduction and chemotaxis in response to IL-8 (9Ben-Baruch A. Xu L. Young P.R. Bengali K. Oppenheim J.J. Wang J.M. J. Biol. Chem. 1995; 270: 22123-22128Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar).In the present study, we show that CXCR2 mutants exhibiting a loss of residues 331–355 (331T) or 342–355 (342T) no longer undergo ligand-enhanced receptor phosphorylation or desensitization as monitored by Ca2+ mobilization in response to ligand challenge. In contrast, receptors exhibiting deletion of amino acid residues 352–355 (352T) continue to undergo phosphorylation in response to ligand. In an effort to determine which specific serine residues are phosphorylated, we mutated individual serine residues 342, 346, 347, and 348 to alanine (S342A, S346A, S347A, S348A) or groups of serine residues to alanine: 346/7/8 (3A) and 342/6/7/8 (4A). Studies with 3ASubE cells expressing these mutant receptors, as compared with 3ASubE cells expressing wild type CXCR2, revealed that serines 342, 346, 347, and 348 of CXCR2 are involved in the desensitization of the receptor to its ligand. However, when these serine residues are mutated to alanine (4A or 3A CXCR2), other serine residues in the carboxyl-terminal domain can be phosphorylated, and the receptor is partially desensitized in response to ligand. In 3ASubE cells, phosphorylation and desensitization of WT CXCR2 occurs within 1 min after ligand treatment, while receptor sequestration requires >30 min. The 342T CXCR2 receptor is not sequestered in response to ligand. These data demonstrate that for CXCR2, specific residues along the carboxyl-terminal domain of the receptor mediate receptor desensitization and sequestration in response to ligand.
Mueller et al. (Sat,) studied this question.