Ludwig, Andreas;
Ehlert, Jan E.;
Flad, Hans-Dieter;
Brandt, Ernst
Identification of Distinct Surface-Expressed and Intracellular CXC-Chemokine Receptor 2 Glycoforms in Neutrophils:N-Glycosylation Is Essential for Maintenance of Receptor Surface Expression
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Media type:
E-Article
Title:
Identification of Distinct Surface-Expressed and Intracellular CXC-Chemokine Receptor 2 Glycoforms in Neutrophils:N-Glycosylation Is Essential for Maintenance of Receptor Surface Expression
Contributor:
Ludwig, Andreas;
Ehlert, Jan E.;
Flad, Hans-Dieter;
Brandt, Ernst
Published:
The American Association of Immunologists, 2000
Published in:
The Journal of Immunology, 165 (2000) 2, Seite 1044-1052
Language:
English
DOI:
10.4049/jimmunol.165.2.1044
ISSN:
0022-1767;
1550-6606
Origination:
Footnote:
Description:
AbstractThe G protein-coupled CXC-chemokine receptor CXCR-2 mediates activation of neutrophil effector functions in response to multiple ligands, including IL-8 and neutrophil-activating peptide 2 (NAP-2). Although CXCR-2 has been successfully cloned and expressed in several cell lines, the molecular properties of the native neutrophil-expressed receptor have remained largely undefined. Here we report on the identification and characterization of distinct CXCR-2 glycoforms and their subcellular distribution in neutrophils. Immunoprecipitation and Western blot analyses of surface-expressed receptors covalently linked to IL-8 or NAP-2 as well as in their unloaded state revealed the occurrence of a single CXCR-2 variant with an apparent size of 56 kDa. According to deglycosylation experiments surface-expressed CXCR-2 carries two N-linked 9-kDa carbohydrate moieties that are both of complex structure. In addition, two other CXCR-2 variants of 38 and 40 kDa were found to occur exclusively intracellular and to carry N-glycosylations of high mannose or hybrid type. These receptors did not participate in ligand-induced receptor trafficking, while surface-expressed CXCR-2 was internalized and re-expressed following stimulation with NAP-2. By enzymatic removal of one 9-kDa carbohydrate moiety in surface-expressed CXCR-2 we can show that neither NAP-2-induced trafficking nor signaling of the receptor is dependent on its full glycosylation. Instead, glycosylation was found to protect CXCR-2 from proteolytic attack, as even partial deglycosylation is associated with serine protease-mediated disappearance of the receptor from the neutrophil surface. Thus, although not directly involved in signaling, glycosylation appears to be required to maintain neutrophil responsiveness to CXC-chemokines during inflammation.