Identification of Distinct Surface-Expressed and Intracellular CXC-Chemokine Receptor 2 Glycoforms in Neutrophils: N-Glycosylation Is Essential for Maintenance of Receptor Surface Expression

• Published in: The Journal of immunology (1950) Vol. 165; no. 2; pp. 1044 - 1052
• Main Authors: Ludwig, Andreas, Ehlert, Jan E, Flad, Hans-Dieter, Brandt, Ernst
• Format: Journal Article
• Language: English
• Published: United States Am Assoc Immnol 15-07-2000
• Subjects: 3T3 Cells; Amidohydrolases - metabolism; Animals; beta-Thromboglobulin; Cell Degranulation - immunology; Cell Membrane - enzymology; Cell Membrane - immunology; Cell Membrane - metabolism; CXCR2 protein; Glycoproteins - biosynthesis; Glycoproteins - metabolism; Glycoproteins - physiology; Glycosylation; Humans; Interleukin-8 - metabolism; Intracellular Fluid - enzymology; Intracellular Fluid - immunology; Intracellular Fluid - metabolism; Ligands; Lysosomes - enzymology; Lysosomes - metabolism; Mice; Molecular Weight; neutrophil-activating peptide 2; Neutrophils - enzymology; Neutrophils - immunology; Neutrophils - metabolism; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; Peptides - metabolism; Protein Isoforms - biosynthesis; Protein Isoforms - metabolism; Protein Isoforms - physiology; Receptors, Chemokine - biosynthesis; Receptors, Chemokine - metabolism; Receptors, Chemokine - physiology; Receptors, Interleukin - biosynthesis; Receptors, Interleukin - metabolism; Receptors, Interleukin - physiology; Receptors, Interleukin-8B; Signal Transduction - immunology
• ISSN: 0022-1767; 1550-6606
• DOI: 10.4049/jimmunol.165.2.1044

The 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.