Skip to main content

Advertisement

SpringerLink
Log in

Adenovirus mediated expression “in vivo” of the chemokine receptor CXCR1

  • Published:
Journal of Structural and Functional Genomics

Abstract

A major hurdle in the structural analysis of membrane proteins is the expression of a functional and homogeneous form of the protein. Except for rhodopsin, most G protein-coupled receptors (GPCRs) are endogenously expressed at very low levels. Heterologous expression of GPCRs in bacteria, yeast, insect cells or mammalian cell lines often yields proteins with large amounts of misfolded proteins and heterogeneous posttranslational modifications. Here, we report a novel mammalian “in vivo” system for the expression of the chemokine receptor CXCR1. This receptor was expressed in liver of mice infected with adenovirus encoding CXCR1. Liver plasma membranes from infected mice displayed high-levels of 125I-labeled human interleukin-8 (IL-8) binding. The pharmacological profile of the recombinant CXCR1 expressed “in vivo” was similar to those expressed in neutrophils. We found that the incorporation of the detergent solubilized CXCR1 into phospholipid vesicles in the presence of Gi/Go proteins is required for the reconstitution of 125I-IL-8 binding. On the basis of the presence of the several endogenous His residues and glycosylation moieties in CXCR1 we fractionated the detergent-solubilized plasma membranes by employing Ni- and Concanavalin A-based chromatography. Fractions enriched with CXCR1 were monitored by 125I-IL-8-bound to the receptor and Western blots with anti-CXCR1 antibodies. This robust expression system could be readily applied for the expression of GPCRs and other eukaryotic membrane proteins.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

DDM:

n-Dodecyl-β-d-maltoside

GFP:

Green fluorescence protein

CMV:

Cytomegalovirus

IL-8:

Interleukin-8

CMC:

Critical micellar concentration

GCP-2:

Human granulocyte chemotactic protein 2

NAP-2:

Neutrophil-activating peptide-2

MGSA:

Melanoma growth-stimulating activity

References

  1. Cherezov V, Rosenbaum DM, Hanson MA, Rasmussen SG, Thian FS, Kobilka TS, Choi HJ, Kuhn P, Weis WI, Kobilka BK, Stevens RC (2007) High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor. Science 318:1258–1265. doi:10.1126/science.1150577

    Article  PubMed  CAS  Google Scholar 

  2. Dowell SJ, Brown AJ (2002) Yeast assays for G-protein-coupled receptors. Recept Channels 8:343–352. doi:10.1080/10606820214647

    Article  PubMed  CAS  Google Scholar 

  3. Eroglu C, Cronet P, Panneels V, Beaufils P, Sinning I (2002) Functional reconstitution of purified metabotropic glutamate receptor expressed in the fly eye. EMBO Rep 3:491–496. doi:10.1093/embo-reports/kvf088

    Article  PubMed  CAS  Google Scholar 

  4. Grisshammer R, Averbeck P, Sohal AK (1999) Improved purification of a rat neurotensin receptor expressed in Escherichia coli. Biochem Soc Trans 27:899–903

    PubMed  CAS  Google Scholar 

  5. Ishida T, Li W, Liu Z, Kiwada H (2006) Stimulatory effect of polyethylene glycol (PEG) on gene expression in mouse liver following hydrodynamics-based transfection. J Gene Med 8:324–334. doi:10.1002/jgm.850

    Article  PubMed  CAS  Google Scholar 

  6. Kypreos KE, van Dijk KW, van Der Zee A, Havekes LM, Zannis VI (2001) Domains of apolipoprotein E contributing to triglyceride and cholesterol homeostasis in vivo. Carboxy l-terminal region 203–299 promotes hepatic very low density lipoprotein triglyceride secretion. J Biol Chem 276:19778–19786. doi:10.1074/jbc.M100418200

    Article  PubMed  CAS  Google Scholar 

  7. LaRosa GJ, Thomas KM, Kaufmann ME, Mark R, White M, Taylor L, Gray G, Witt D, Navarro J (1992) Amino terminus of the interleukin-8 receptor is a major determinant of receptor subtype specificity. J Biol Chem 267:25402–25406

    PubMed  CAS  Google Scholar 

  8. Loisel TP, Ansanay H, St-Onge S, Gay B, Boulanger P, Strosberg AD, Marullo S, Bouvier M (1997) Recovery of homogeneous and functional beta 2-adrenergic receptors from extracellular baculovirus particles. Nat Biotechnol 15:1300–1304. doi:10.1038/nbt1197-1300

    Article  PubMed  CAS  Google Scholar 

  9. Mirzabekov T, Bannert N, Farzan M, Hofmann W, Kolchinsky P, Wu L, Wyatt R, Sodroski J (1999) Enhanced expression, native purification, and characterization of CCR5, a principal HIV-1 coreceptor. J Biol Chem 274:28745–28750. doi:10.1074/jbc.274.40.28745

    Article  PubMed  CAS  Google Scholar 

  10. Okada T, Fujiyoshi Y, Silow M, Navarro J, Landau EM, Shichida Y (2002) Functional role of internal water molecules in rhodopsin revealed by X-ray crystallography. Proc Natl Acad Sci USA 99:5982–5987. doi:10.1073/pnas.082666399

    Article  PubMed  CAS  Google Scholar 

  11. Oprian DD, Molday RS, Kaufman RJ, Khorana HG (1987) Expression of a synthetic bovine rhodopsin gene in monkey kidney cells. Proc Natl Acad Sci USA 84:8874–8878. doi:10.1073/pnas.84.24.8874

    Article  PubMed  CAS  Google Scholar 

  12. Palczewski K, Kumasaka T, Hori T, Behnke CA, Motoshima H, Fox BA, Le Trong I, Teller DC, Okada T, Stenkamp RE, Yamamoto M, Miyano M (2000) Crystal structure of rhodopsin: a G protein-coupled receptor. Science 289:739–745. doi:10.1126/science.289.5480.739

    Article  PubMed  CAS  Google Scholar 

  13. Prado GN, Suetomi K, Shumate D, Maxwell C, Ravindran A, Rajarathnam K, Navarro J (2007) Chemokine signaling specificity: essential role for the N-terminal domain of chemokine receptors. Biochemistry 46:8961–8968. doi:10.1021/bi7004043

    Article  PubMed  CAS  Google Scholar 

  14. Rasmussen SG, Choi HJ, Rosenbaum DM, Kobilka TS, Thian FS, Edwards PC, Burghammer M, Ratnala VR, Sanishvili R, Fischetti RF, Schertler GF, Weis WI, Kobilka BK (2007) Crystal structure of the human beta2 adrenergic G-protein-coupled receptor. Nature 450:383–387. doi:10.1038/nature06325

    Article  PubMed  CAS  Google Scholar 

  15. Reeves PJ, Kim JM, Khorana HG (2002) Structure and function in rhodopsin: a tetracycline-inducible system in stable mammalian cell lines for high-level expression of opsin mutants. Proc Natl Acad Sci USA 99:13413–13418. doi:10.1073/pnas.212519199

    Article  PubMed  CAS  Google Scholar 

  16. Reeves PJ, Callewaert N, Contreras R, Khorana HG (2002) Structure and function in rhodopsin: high-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N-acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line. Proc Natl Acad Sci USA 99:13419–13424. doi:10.1073/pnas.212519299

    Article  PubMed  CAS  Google Scholar 

  17. Ruot B, Breuille D, Rambourdin F, Bayle G, Capitan P, Obled C (2000) Synthesis rate of plasma albumin is a good indicator of liver albumin synthesis in sepsis. Am J Physiol Endocrinol Metab 279:E244–E251

    PubMed  CAS  Google Scholar 

  18. Sarkar CA, Dodevski I, Kenig M, Dudli S, Mohr A, Hermans E, Plückthun A (2008) Directed evolution of a G protein-coupled receptor for expression, stability, and binding selectivity. Proc Natl Acad Sci USA 105:14808–14813. doi:10.1073/pnas.0803103105

    Article  PubMed  CAS  Google Scholar 

  19. Sarramegna V, Talmont F, Demange P, Milon A (2003) Heterologous expression of G-protein-coupled receptors: comparison of expression systems fron the standpoint of large-scale production and purification. Cell Mol Life Sci 60:1529–1546. doi:10.1007/s00018-003-3168-7

    Article  PubMed  CAS  Google Scholar 

  20. Sternweis PC, Robishaw JD (1984) Isolation of two proteins with high affinity for guanine nucleotides from membranes of bovine brain. J Biol Chem 259:13806–13813

    PubMed  CAS  Google Scholar 

  21. Suzuki H, Prado GN, Wilkinson N, Navarro J (1994) The N terminus of interleukin-8 (IL-8) receptor confers high affinity binding to human IL-8. J Biol Chem 269:18263–18266

    PubMed  CAS  Google Scholar 

  22. Telford JN, Langworthy TA, Racker E (1984) Three proton pumps, morphology and movements. J Bioenerg Biomembr 16:335–351. doi:10.1007/BF00743230

    Article  PubMed  CAS  Google Scholar 

  23. Thomas KM, Taylor L, Navarro J (1991) The interleukin-8 receptor is encoded by a neutrophil-specific cDNA clone, F3R. J Biol Chem 266:14839–14841

    PubMed  CAS  Google Scholar 

  24. Thomas KM, Taylor L, Prado G, Romero J, Moser B, Car B, Walz A, Baggiolini M, Navarro J (1994) Functional and ligand binding specificity of the rabbit neutrophil IL-8 receptor. J Immunol 152:2496–2500

    PubMed  CAS  Google Scholar 

  25. Viola A, Luster AD (2008) Chemokines and their receptors: drug targets in immunity and inflammation. Annu Rev Pharmacol Toxicol 48:171–197. doi:10.1146/annurev.pharmtox.48.121806.154841

    Article  PubMed  CAS  Google Scholar 

  26. Warne T, Serrano-Vega MJ, Baker JG, Moukhametzianov R, Edwards PC, Henderson R, Leslie AG, Tate CG, Schertler GF (2008) Structure of a beta1-adrenergic G-protein-coupled receptor. Nature 454:486–491. doi:10.1038/nature07101

    Article  PubMed  CAS  Google Scholar 

  27. Williamson K, Dickey BF, Pyun HY, Navarro J (1988) Solubilization and reconstitution of the formylmethionylleucylphenylalanine receptor coupled to guanine nucleotide regulatory protein. Biochemistry 27:5371–5377. doi:10.1021/bi00414a062

    Article  PubMed  CAS  Google Scholar 

  28. Yang M, Baranov E, Moossa AR, Penman S, Hoffman RM (2000) Visualizing gene expression by whole-body fluorescence imaging. Proc Natl Acad Sci USA 97:12278–12282. doi:10.1073/pnas.97.22.12278

    Article  PubMed  CAS  Google Scholar 

  29. Zhang L, Salom D, He J, Okun A, Ballesteros J, Palczewski K, Li N (2005) Expression of functional G protein-coupled receptors in photoreceptors of transgenic Xenopus laevis. Biochemistry 44:14509–14518. doi:10.1021/bi051386z

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We are grateful to Professor David Konkel of our Department of Biochemistry and Molecular Biology for editing and valuable discussions. This work was supported by the National Institutes of Health R01 GM081798 and the Welch Foundation.

Author information

Authors and Affiliations

  1. Department of Neuroscience and Cell Biology, University of Texas Medical Branch, 301 University Blvd., Galveston, TX, 77555, USA

    J. Sarmiento, G. N. Prado, K. Suetomi, C. Stanzel, C. Maxwell, D. Shumate, M. R. Tandang-Silvas & J. Navarro

  2. Sealy Centers of Molecular Medicine and Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX, 77555, USA

    J. Sarmiento, G. N. Prado, K. Suetomi, C. Stanzel, C. Maxwell, D. Shumate, M. R. Tandang-Silvas, K. Rajarathnam & J. Navarro

  3. Pharmacology Unit, University of Patras Medical School, Panepistimioupolis Rio, TK, 26500, Greece

    K. E. Kypreos

  4. Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, 77555, USA

    K. Rajarathnam

Authors
  1. J. Sarmiento
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. K. E. Kypreos
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. G. N. Prado
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. K. Suetomi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. C. Stanzel
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. C. Maxwell
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. D. Shumate
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. M. R. Tandang-Silvas
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. K. Rajarathnam
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. J. Navarro
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to J. Navarro.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sarmiento, J., Kypreos, K.E., Prado, G.N. et al. Adenovirus mediated expression “in vivo” of the chemokine receptor CXCR1. J Struct Funct Genomics 10, 17–23 (2009). https://doi.org/10.1007/s10969-008-9051-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10969-008-9051-x

Keywords

Search

Navigation