Functional Expression of CXCR4 in S. cerevisiae: Development of Tools for Mechanistic and Pharmacologic Studies

  • W.-B. Zhang
  • Z.-X. Wang
  • J. L. Murray
  • N. Fujii
  • J. Broach
  • S. C. Peiper
Conference paper
Part of the Ernst Schering Research Foundation Workshop book series (SCHERING FOUND, volume 45)


Chemokines are cytokines that program directed migration of leukocyte subsets (Loetscher et al. 2000). These small proteins (~8–14 kDa) form the largest family of secreted intercellular messengers, having over 43 members recognized at this time. The family is characterized by the presence of four positionally conserved cysteine residues and subdivided into four branches based on the relationship of the two amino-proximal cysteines: C-C, C-X-C, C-X3-C, and C. Cellular signals for chemokines are transduced by members of the G-protein coupled receptor (GPCR) family that also cosegregate along these four subdivisions (Murphy et al. 2000). It is now recognized that in addition to promoting inflammation through recruitment of leukocyte subsets, chemokines play a central role in directing the development of tissues outside the hemato-lymphoid system.


Yeast Strain Reporter Gene Expression CXCR4 Antagonist Tyrosine Sulfation Pheromone Response Pathway 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


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  1. Baranski TJ, Herzmark P, Lichtarge O, Gerber BO, Trueheart J, Meng EC, Iiri T, Sheikh SP, Bourne HR (1999) C5a receptor activation. Genetic identification of critical residues in four transmembrane helices. J Biol Chem 274:15757–15765PubMedCrossRefGoogle Scholar
  2. Bleul CC, Farzan M, Choe H, Parolin C, Clark-Lewis I, Sodroski J, Springer TA (1996) The lymphocyte chemoattractant SDF-1 is a ligand for LESTR/fusin and blocks HIV- 1 entry. Nature 382:829–833PubMedCrossRefGoogle Scholar
  3. Boshoff C, Endo Y, Collins PD, Takeuchi Y, Reeves JD, Schweickart VL, Siani MA, Sasaki T, Williams TJ, Gray PW, Moore PS, Chang Y, Weiss RA (1997) Angiogenic and HIV- inhibitory functions of KSHV- encoded chemokines. Science 278:290–294PubMedCrossRefGoogle Scholar
  4. Brelot A, Heveker N, Montes M, Alizon M (2000) Identification of residues of CXCR4 critical for human immunodeficiency virus coreceptor and chemokine receptor activities. J Biol Chem 275:23736–23744PubMedCrossRefGoogle Scholar
  5. Chabot DJ, Chen H, Dimitrov DS, Broder CC (2000) N-linked glycosylation of CXCR4 masks coreceptor function for CCRS-dependent human immunodeficiency virus type 1 isolates. J Virol 74:4404–4413PubMedCrossRefGoogle Scholar
  6. Erickson JR, Wu JJ, Goddard JG, Tigyi G, Kawanishi K, Tomei LD, Kiefer MC (1998) Edg-2/Vzg-1 couples to the yeast pheromone response pathway selectively in response to lysophosphatidic acid. J Biol Chem 273:1506–1510PubMedCrossRefGoogle Scholar
  7. Farrens DL, Altenbach C, Yang K, Hubbell WL, Khorana HG (1996) Requirement of rigid-body motion of transmembrane helices for light activation of rhodopsin. Science 274:768–770PubMedCrossRefGoogle Scholar
  8. Farzan M., Babcock GJ, Vasilieva N, Wright PL, Kiprilov E, Mirzabekov T, Choe H (2002) The role of post-translational modifications of the CXCR4 amino terminus in stromal-derived factor 1 a association and HIV 1 entry. J Biol Chem 277:29484–29489PubMedCrossRefGoogle Scholar
  9. Feng Y, Broder CC, Kennedy PE, Berger, EA (1996) HIV- 1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science 272:872–877PubMedCrossRefGoogle Scholar
  10. Gether U, Lin S, Ghanouni P, Ballesteros JA, Weinstein H, Kobilka BK (1997) Agonists induce conformational changes in transmembrane domains III and VI of the β2 adrenoceptor. EMBO J 16:6737–6747PubMedCrossRefGoogle Scholar
  11. Geva A, Lassere TB, Lichtarge O, Pollitt SK, Baranski TJ (2000) Genetic mapping of the human C5a receptor. Identification of transmembrane amino acids critical for receptor function. J Biol Chem 275:35393–35401PubMedCrossRefGoogle Scholar
  12. Ho HH, Ganeshalingam N, Rosenhouse-Dantsker A, Osman R, Gershengorn MC (2001) Charged residues at the intracellular boundary of transmembrane helices 2 and 3 independently affect constitutive activity of Kaposi’s sarcoma-associated herpesvirus G protein-coupled receptor. J Biol Chem 276:1376–1382PubMedCrossRefGoogle Scholar
  13. Hu QX, Barry AP, Wang ZX, Connolly SM, Peiper SC, Greenberg ML (2000) Evolution of the human immunodeficiency virus type 1 envelope during infection reveals molecular corollaries of specificity for coreceptor utilization and AIDS pathogenesis. J Virol 74:11858–11872PubMedCrossRefGoogle Scholar
  14. King K, Dohlman HG, Thorner J, Caron MG, Lefkowitz RJ (1990) Control of yeast mating signal transduction by a mammalian β2-adrenergic receptor and Gs a subunit. Science 250:121–123PubMedCrossRefGoogle Scholar
  15. Klein C, Paul JI, Sauve K, Schmidt MM, Arcangeli L, Ransom J, Trueheart J, Manfredi JP, Broach JR, Murphy AJ (1998) Identification of surrogate agonists for the human FPRL-1 receptor by autocrine selection in yeast. Nat Biotechnol 16:1334–1337PubMedCrossRefGoogle Scholar
  16. Lin SW, Sakmar TP (1996) Specific tryptophan UV- absorbance changes are probes of the transition of rhodopsin to its active state. Biochemistry 35:11149–11159PubMedCrossRefGoogle Scholar
  17. Loetscher P, Moser B, Baggiolini M (2000) Chemokines and their receptors in lymphocyte traffic and HIV infection. Adv Immunol 74:127–180PubMedCrossRefGoogle Scholar
  18. Lu Z, Berson JF, Chen Y, Turner JD, Zhang T, Sharron M, Jenks MH, Wang Z, Kim J, Rucker J, Hoxie JA, Peiper SC, Doms RW (1997) Evolution of HIV- 1 coreceptor usage through interactions with distinct CCR5 and CXCR4 domains. Proc Natl Acad Sci U S A 94:6426–6431PubMedCrossRefGoogle Scholar
  19. Ma Q, Jones D, Borghesani PR, Segal RA, Nagasawa T, Kishimoto T, Bronson RT, Springer TA (1998) Impaired B-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4- and SDF-1-deficient mice. Proc Natl Acad Sci U S A 95:9448–9453PubMedCrossRefGoogle Scholar
  20. Muller A, Homey B, Soto H, Ge N, Catron D, Buchanan ME, McClanahan T, Murphy E, Yuan W, Wagner SN, Barrera JL, Mohar A, Verastegui E, Zlotnik A (2001) Involvement of chemokine receptors in breast cancer metastasis. Nature 410:50–56PubMedCrossRefGoogle Scholar
  21. Murakami T, Nakajima T, Koyanagi Y, Tachibana K, Fujii N, Tamamura H, Yoshida N, Waki M, Matsumoto A, Yoshie O, Kishimoto T, Yamamoto N, Nagasawa T (1997) A small molecule CXCR4 inhibitor that blocks T cell line-tropic HIV- 1 infection. J Exp Med 186:1389–1393PubMedCrossRefGoogle Scholar
  22. Murphy PM, Baggiolini M, Charo IF, Hebert CA, Horuk R, Matsushima K, Miller LH, Oppenheim JJ, Power CA (2000) International union of pharmacology. XXII. Nomenclature for chemokine receptors. Pharmacol Rev 52:145–176PubMedGoogle Scholar
  23. Nagasawa T, Hirota S, Tachibana K, Takakura N, Nishikawa S, Kitamura Y, Yoshida N, Kikutani H, Kishimoto T (1996) Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1. Nature 382:635–638PubMedCrossRefGoogle Scholar
  24. Navenot JM, Wang ZX, Trent JO, Murray JL, Hu QX, DeLeeuw L, Moore PS, Chang Y, Peiper SC (2001) Molecular anatomy of CCR5 engagement by physiologic and viral chemokines and HIV- 1 envelope glycoproteins: differences in primary structural requirements for RANTES, MIP-1 a, and vMIP-II binding. J Mol Biol 313:1181–1193PubMedCrossRefGoogle Scholar
  25. Niehrs C, Huttner WB, Carvallo D, Degryse E (1990) Conversion of recombinant hirudin to the natural form by in vitro tyrosine sulfation. Differential substrate specificities of leech and bovine tyrosylprotein sulfotransferases. J Biol Chem 265:9314–9318PubMedGoogle Scholar
  26. Oberlin E, Amara A, Bachelerie F, Bessia C, Virelizier JL, Arenzana-Seisdedos F, Schwartz O, Heard JM, Clark-Lewis I, Legler DF, Loetscher M, Baggiolini M, Moser B (1996) The CXC chemokine SDF-1 is the ligand for LESTR/fusin and prevents infection by T- cell-line-adapted HIV- 1. Nature 382:833–835PubMedCrossRefGoogle Scholar
  27. Pauwels PJ, Wurch T (1998) Review: amino acid domains involved in constitutive activation of G-protein-coupled receptors. Mol Neurobiol 17:109–135PubMedCrossRefGoogle Scholar
  28. Price LA, Kajkowski EM, Hadcock JR, Ozenberger BA, Pausch MH (1995) Functional coupling of a mammalian somatostatin receptor to the yeast pheromone response pathway. Mol Cell Biol 15:6188–6195PubMedGoogle Scholar
  29. Samama P, Cotecchia S, Costa T, Lefkowitz RJ (1993) A mutation-induced activated state of the β2-adrenergic receptor. Extending the ternary complex model. J Biol Chem 268:4625–4636PubMedGoogle Scholar
  30. Schols D, Struyf S, Van Damme J, Este JA, Henson G, De Clercq E (1997) Inhibition of T- tropic HIV strains by selective antagonization of the chemokine receptor CXCR4. J Exp Med 186:1383–1388PubMedCrossRefGoogle Scholar
  31. Sheikh SP, Zvyaga TA, Lichtarge O, Sakmar TP, Bourne HR (1996) Rhodopsin activation blocked by metal-ion-binding sites linking transmembrane helices C and F. Nature 383:347–350PubMedCrossRefGoogle Scholar
  32. Tachibana K, Hirota S, Iizasa H, Yoshida H, Kawabata K, Kataoka Y, Kitamura Y, Matsushima K, Yoshida N, Nishikawa S, Kishimoto T, Nagasawa T (1998) The chemokine receptor CXCR4 is essential for vascularization of the gastrointestinal tract. Nature 393:591–594PubMedCrossRefGoogle Scholar
  33. Tamamura H, Omagari A, Oishi S, Kanamoto T, Yamamoto N, Peiper SC, Nakashima H, Otaka A, Fujii N (2000) Pharmacophore identification of a specific CXCR4 inhibitor, T140, leads to development of effective antiHIV agents with very high selectivity indexes. Bioorg Med Chem Lett 10:2633–2637PubMedCrossRefGoogle Scholar
  34. Willett BJ, Adema K, Heveker N, Brelot A, Picard L, Alizon M, Turner JD, Hoxie JA, Peiper S, Neil JC, Hosie MJ (1998) The second extracellular loop of CXCR4 determines its function as a receptor for feline immunodeficiency virus. J Virol 72:6475–6481PubMedGoogle Scholar
  35. Xu H, Petersen EI, Petersen SB, el-Gewely MR (1999) Random mutagenesis libraries: optimization and simplification by PCR. Biotechniques 27:1102–1104, 1106, 1108PubMedGoogle Scholar
  36. Zhang WB, Navenot JM, Haribabu B, Tamamura H, Hiramatu K, Omagari A, Pei G, Manfredi JP, Fujii N, Broach JR, Peiper SC (2002) A point mutation that confers constitutive activity to CXCR4 reveals that T140 is an inverse agonist and that AMD3100 and ALX40–4C are weak partial agonists. J Biol Chem 277:24515–24521PubMedCrossRefGoogle Scholar
  37. Zhou H, Tai HH (1999) Characterization of recombinant human CXCR4 in insect cells: role of extracellular domains and N-glycosylation in ligand binding. Arch Biochem Biophys 369:267–276PubMedCrossRefGoogle Scholar
  38. Zou YR, Kottmann AH, Kuroda M, Taniuchi I, Littman DR (1998) Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development. Nature 393:595–599PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2004

Authors and Affiliations

  • W.-B. Zhang
  • Z.-X. Wang
  • J. L. Murray
  • N. Fujii
  • J. Broach
  • S. C. Peiper

There are no affiliations available

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