Normal Cell Surface Expression and Selective Loss of Functions Resulting from Phe110 to Ser and Cys126 to Trp Substitutions in the Formyl Peptide Receptor

Abstract

The N‐formyl peptide receptor (FPR) is a G protein‐coupled chemoattractant receptor that mediates diverse leukocyte functions when stimulated by bacteria‐derived N‐formyl peptides such as fMet‐Leu‐Phe (fMLF). Impaired neutrophil responsiveness to fMLF parallels increased susceptibility to periodontal microorganisms among patients with localized juvenile periodontitis (LJP). To determine whether the recently identified FPR mutations in LJP patients are responsible for selective loss of receptor‐mediated functions, we prepared and analyzed RBL‐2H3 cells expressing FPR bearing Phe110 to Ser (FPR‐F110S) or Cys‐126 to Trp (FPR‐C126W) replacement as well as a FPR double mutant (FPR‐FSCW). All mutant receptors were expressed normally on the cell surface, but were unable to mediate release of β‐hexosaminidase upon fMLF stimulation. FPR‐C126W effectively mediated fMLF uptake, an indication of receptor‐mediated endocytosis, whereas FPR‐F110S and FSCW exhibited markedly reduced ability to uptake fMLF. Both FPR‐F110S and FPR‐C126W were defective in chemotaxis and displayed reduced Ca^{2 +} mobilization, but mutation at both positions partially restored the ability to respond to fMLF in chemotaxis assay and was nearly normal in Ca^{2 +} mobilization assay. All mutants exhibited diminished accumulation of inositol phosphates. FPR‐F110S displayed a delayed and significantly reduced ERK phosphorylation whereas FPR‐FSCW nearly lost the ability to phosphorylate ERK. Taken together, these results indicate compromised signaling capabilities due to the FPR mutations, but the loss of function is selective and could be partially rescued by mutations at both positions.

• Neutrophils
• Inflammation
• Chemotaxis
• Cellular activation

Abbreviations
FPR:	=	formyl peptide receptor
fMLF:	=	N‐formyl‐Met‐Leu‐Phe
LJP:	=	localized juvenile periodontitis
G protein:	=	guanine nucleotide‐binding regulatory protein
GPCR:	=	G protein‐coupled receptor
PTX:	=	pertussis toxin
ERK:	=	extracellular signal‐regulated protein kinase
PLCβ:	=	phospholipase C‐β