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

  • J. Sarmiento
  • K. E. Kypreos
  • G. N. Prado
  • K. Suetomi
  • C. Stanzel
  • C. Maxwell
  • D. Shumate
  • M. R. Tandang-Silvas
  • K. Rajarathnam
  • J. Navarro
Article

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.

Keywords

Chemokines GPCR Adenovirus Receptors Reconstitution Protein expression 

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

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Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • J. Sarmiento
    • 1
    • 2
  • K. E. Kypreos
    • 3
  • G. N. Prado
    • 1
    • 2
  • K. Suetomi
    • 1
    • 2
  • C. Stanzel
    • 1
    • 2
  • C. Maxwell
    • 1
    • 2
  • D. Shumate
    • 1
    • 2
  • M. R. Tandang-Silvas
    • 1
    • 2
  • K. Rajarathnam
    • 2
    • 4
  • J. Navarro
    • 1
    • 2
  1. 1.Department of Neuroscience and Cell BiologyUniversity of Texas Medical BranchGalvestonUSA
  2. 2.Sealy Centers of Molecular Medicine and Structural Biology and Molecular BiophysicsUniversity of Texas Medical BranchGalvestonUSA
  3. 3.Pharmacology UnitUniversity of Patras Medical SchoolPanepistimioupolis RioGreece
  4. 4.Department of Biochemistry and Molecular BiologyUniversity of Texas Medical BranchGalvestonUSA

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