Critical role of N-terminal N-glycosylation for proper folding of the human formyl peptide receptor.
The human formyl peptide receptor (FPR) is N-glycosylated and activates phagocytes via G(i)-proteins. The FPR expressed with G(i)alpha(2)beta(1)gamma(2) in Sf9 insect cells exhibits high constitutive activity as assessed by strong inhibitory effects of an inverse agonist and Na(+) on basal guanosine 5(')-O-(3-thiotriphosphate) (GTPgammaS) binding. The aim of our study was to analyze the role of N-glycosylation in FPR function. Site-directed mutagenesis of extracellular Asn residues prevented FPR glycosylation but not FPR expression in Sf9 membranes. However, in terms of high-affinity agonist binding, kinetics of GTPgammaS binding, number of G(i)-proteins activated, and constitutive activity, non-glycosylated FPR was much less active than native FPR. FPR-Asn4Gln/Asn10Gln/Asn179Gln and FPR-Asn4Gln/Asn10/Gln exhibited similar defects. Our data indicate that N-glycosylation of N-terminal Asn4 and Asn10 but not of Asn179 in the second extracellular loop is essential for proper folding and, hence, function of FPR. FPR deglycosylation by bacterial glycosidases could be a mechanism by which bacteria compromise host defense.
Wenzel-Seifert
Katharina
K
Department of Pharmacology and Toxicology, The University of Kansas, Lawrence, KS 66045-7582, USA.
Seifert
Roland
R
D001216:Asparagine / Q000378:metabolism; D001419:Bacteria / Q000472:pathogenicity; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D006031:Glycosylation; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D009154:Mutation; D017510:Protein Folding; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018000:Receptors, Peptide / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012964:Sodium / Q000494:pharmacology; D014415:Tunicamycin / Q000494:pharmacology
D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D018000:Receptors, Peptide; D014415:Tunicamycin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D001216:Asparagine; D012964:Sodium