Abstract
The gene encoding the human formyl peptide receptor 1 (FPR1) is heterogeneous, containing numerous single nucleotide polymorphisms (SNPs). Here, we examine the effect of these SNPs on gene transcription and protein translation. We also identify gene promoter sequences and putative FPR1 transcription factors. To test the effect of codon bias and codon pair bias on FPR1 expression, four FPR1 genetic variants were expressed in human myeloid U937 cells fused to a reporter gene encoding firefly luciferase. No significant differences in luciferase activity were detected, suggesting that the translational regulation and protein stability of FPR1 are modulated by factors other than the SNP codon bias and the variant amino acid properties. Deletion and mutagenesis analysis of the FPR1 promoter showed that a CCAAT box is not required for gene transcription. A −88/41 promoter construct resulted in the strongest transcriptional activity, whereas a −72/41 construct showed large reduction in activity. The region between −88 and −72 contains a consensus binding site for the transcription factor PU.1. Mutagenesis of this site caused significant reduction in reporter gene expression. The PU.1 binding was confirmed in vivo by chromatin immunoprecipitation, and the binding to nucleotides −84 to −76 (TTCCTATTT) was confirmed in vitro by an electrophoretic mobility shift assay. Thus, similar to many other myeloid genes, FPR1 promoter activity requires PU.1. Two single nucleotide polymorphisms at −56 and −54 did not significantly affect FPR1 gene expression, despite differences in binding of transcription factor IRF1 in vitro. Inflammatory mediators such as interferon-γ, tumor necrosis factor-α, and lipopolysaccharide did not increase FPR1 promoter activity in myeloid cells, whereas differentiation induced by DMSO and retinoic acid enhanced the activity. This implies that the expression of FPR1 in myeloid cells is developmentally regulated, and that the differentiated cells are equipped for immediate response to microbial infections.
Highlights
Formyl peptide receptor 1 (FPR1) is a G protein-coupled receptor that mediates important host defense functions such as chemotaxis and killing of microorganisms through phagocytosis and oxidative burst [1]
Human myeloid U937 cells were co-transfected with various amounts of these plasmids and a constant amount of the pRL-TK vector which drives the expression of Renilla luciferase under the TK promoter
The codon bias and codon pair bias differences based on the single nucleotide polymorphisms (SNPs) in the coding region of FPR1 do not appear to affect the expression levels of the receptor in transfected U937 cells
Summary
Formyl peptide receptor 1 (FPR1) is a G protein-coupled receptor that mediates important host defense functions such as chemotaxis and killing of microorganisms through phagocytosis and oxidative burst [1]. The coding sequence of FPR1 contains ten single nucleotide polymorphisms (SNPs); six are non-synonymous, resulting in amino acid changes, and four are synonymous [2,3,4]. FPR1, which contains 350 amino acids, could theoretically be encoded in .10183 ways, with each adjacent pair of amino acids encoded by 2–36 different pairs of synonymous codons. Codon pairs are used more or less frequently than expected, but not always following the codon bias frequencies. Based on the codon frequencies mentioned above, the amino acid pair Val-Leu is expected to be encoded by GTG-CTG much more frequently than GTA-TTA, but this sequence is encoded somewhat less frequently by GTG-CTG than by GTA-TTA (codon pair bias scores of 0.144 and 0.397, respectively) (www.sciencemag.org/cgi/ content/full/320/5884/1784/DC1; [6]). The reason for the poor translation efficiency is thought to be certain tRNAs that interact poorly on the ribosomal A- and P-sites of underrepresented codon pairs [7]
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