Critical role of N-terminal N-glycosylation for proper folding of the human formyl peptide receptor

Abstract

The human formyl peptide receptor (FPR) is N-glycosylated and activates phagocytes via G i-proteins. The FPR expressed with G iα 2β 1γ 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) (GTPγS) 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 GTPγS 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.