Defective Gi Protein Coupling in Two Formyl Peptide Receptor Mutants Associated with Localized Juvenile Periodontitis

Abstract

Neutrophils play an important role in host defense against bacterial infections and in the pathogenesis of various inflammatory diseases (1, 2). Neutrophils express GPCRs 1 for the chemoattractants fMLP, complement C5a, interleukin-8, leukotriene B 4 , and platelet-activating factor (3-6). Chemoattractant receptors couple to G i proteins to activate phospholipase C␤2 and phosphoinositide 3-kinase-␥, with subsequent stimulation of chemotaxis, oxygen radical formation, and granule release (3, 7-9). Disruption of the FPR gene in mice increases susceptibility to infection by Listeria monocytogenes (10). However, although at cellular and molecular levels, the human FPR is one of the most extensively studied GPCRs (3-6, 8, 11), the in vivo role of the FPR in man has remained elusive.LJP is a defined periodontal disease that begins at ϳ11-15 years of age, affects the permanent incisors and/or first molars, and is associated with the presence of Actinobacillus actinomycetemcomitans in subgingival pockets. LJP is not associated with other diseases, and there is evidence that LJP is caused by a genetic defect (12, 13). It has been known for a long time that neutrophils from LJP patients show reduced binding of the agonist fMLP and reduced fMLP-induced chemotaxis (14 -16). Genetic analysis of 30 LJP patients revealed that 29 of those patients possess a F110S and/or C126W mutation in the FPR gene (17). In contrast, none of 31 patients with adult periodontitis and none of 20 control subjects possess a F110S or C126W mutation (17). The F110S mutation resides in the third transmembrane domain, whereas the C126W mutation resides in the second intracellular loop (Fig. 1). The second intracellular loop of the FPR is important for G i protein coupling (18), and a C126S mutation uncouples the FPR from G i proteins (19). Based on all these findings, we developed the hypothesis that the underlying cause of LJP is defective G i protein coupling of the FPR. To test this hypothesis, we engineered FLAG epitopetagged FPR-F110S and FPR-C126W and analyzed coupling of these FPR mutants and FPR-WT to the G protein G␣ i2 ␤ 1 ␥ 2 using Sf9 insect cells as the expression system. In previous studies, we already showed that Sf9 insect cells are a very sensitive system for studying G i protein coupling of chemoattractant receptors (20 -22). Here we report that FPR-F110S and FPR-C126W show an almost complete defect and a complete defect in G i protein coupling, respectively. The discrete clinical symptoms associated with the defective FPR indicate that the loss of FPR function in host defense is, for the most part, readily compensated. EXPERIMENTAL PROCEDURESMaterials-The baculovirus encoding G␣ i2 was kindly provided by Dr. A. G. Gilman (Department of Pharmacology, University of Southwestern Medical Center, Dallas, TX). Recombinant baculovirus encoding the unmodified versions of the G protein ␤ 1 ␥ 2 subunits was a kind gift of Dr.

Evaluation of human leukocyte N-formylpeptide receptor (FPR1) SNPs in aggressive periodontitis patients

“…This conclusion assumes that the FPR-F110S and FPR-C126W mutations occur and are associated with LJP. The lack of identification of a single FPR-F110S or FPR-C126W SNP in any of the more than 250 individuals (500 chromosomes) tested to date (current study and Sahagun-Ruiz et al 25 ) suggests that these SNPs are rare, if they occur. It is possible that these results are due to population stratification in the AfricanAmerican populations tested.…”

“…25 The authors note that the discrete LJP clinical phenotype contrasts with the severe functional defect for FPR1-F110S and FPR1-C126W. They conclude loss of function in host FPR1 must be readily compensated.…”

Multiple Differences in Agonist and Antagonist Pharmacology between Human and Guinea Pig Histamine H1-Receptor

“…Recombinant gpH 1 R exhibited very different migration in SDS-PAGE than hH 1 R, i.e., we detected faint diffuse ϳ36-and ϳ50-kDa bands and intense crisp ϳ16-and ϳ30-kDa bands in Sf9 membranes expressing gpH 1 R. In contrast to the results obtained with hH 1 R, tunicamycin had no effect on migration of gpH 1 R, pointing to different types of glycosylation in the two H 1 R species isoforms. Currently, we do not know the identity of the multiple bands in Sf9 membranes expressing gpH 1 R, but atypical migration of GPCRs in SDS-PAGE has been repeatedly observed (Grü newald et al, 1996;Kelley et al, 2001;Seifert and Wenzel-Seifert, 2001). Because even complex supramolecular structures such as GPCR dimers are preserved in SDS-PAGE (Fukushima et al, 1997;Hebert and Bouvier, 1998;Kelley et al, 2001), it is possible that the different electrophoretic mobilities of hH 1 R and gpH 1 R reflect different GPCR conformations.…”

Expression and Function of Formyl Peptide Receptors on Human Fibroblast Cells

“…In neutrophils, FPR couples to pertussis toxin-sensitive G␣ i proteins, which activate PLC leading to the breakdown of phosphatidylinositol 4,5-biphosphate to inositol 1,4,5-phosphate resulting in intracellular calcium mobilization (6,22,23). To determine whether ligand binding to FPRs induces similar signal transduction in fibroblast cells, we examined the stimulated release of calcium from intracellular stores.…”