Identification of distinct surface-expressed and intracellular CXC-chemokine receptor 2 glycoforms in neutrophils: N-glycosylation is essential for maintenance of receptor surface expression.
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.
Ludwig
A
A
Department of Immunology, Forschungszentrum Borstel, Borstel, Germany. aludwig@fz-borstel.de
Ehlert
J E
JE
Flad
H D
HD
Brandt
E
E
D016475:3T3 Cells; D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D015550:Cell Degranulation / Q000276:immunology; D002462:Cell Membrane / Q000201:enzymology / Q000276:immunology / Q000378:metabolism; D006023:Glycoproteins / Q000096:biosynthesis* / Q000378:metabolism / Q000502:physiology; D006031:Glycosylation; D006801:Humans; D016209:Interleukin-8 / Q000378:metabolism*; D007424:Intracellular Fluid / Q000201:enzymology / Q000276:immunology / Q000378:metabolism*; D008024:Ligands; D008247:Lysosomes / Q000201:enzymology / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D009504:Neutrophils / Q000201:enzymology / Q000276:immunology / Q000378:metabolism*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010455:Peptides / Q000378:metabolism; D020033:Protein Isoforms / Q000096:biosynthesis / Q000378:metabolism / Q000502:physiology; D019707:Receptors, Chemokine / Q000096:biosynthesis* / Q000378:metabolism / Q000502:physiology; D018123:Receptors, Interleukin / Q000096:biosynthesis* / Q000378:metabolism / Q000502:physiology; D023063:Receptors, Interleukin-8B; D015398:Signal Transduction / Q000276:immunology; D001620:beta-Thromboglobulin
D006023:Glycoproteins; D016209:Interleukin-8; D008024:Ligands; C061615:PPBP protein, human; D010455:Peptides; D020033:Protein Isoforms; D019707:Receptors, Chemokine; D018123:Receptors, Interleukin; D023063:Receptors, Interleukin-8B; D001620:beta-Thromboglobulin; C009862:connective tissue-activating peptide; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase