Abstract

The N-formyl peptide chemoattractant receptor (fMLF-R) is a cell-surface, G-protein-coupled glycoprotein that mediates the directed locomotion of neutrophils upon binding N-formylated peptides. The fMLF-R is encoded primarily by a 1.6-kb mRNA in differentiated HL-60 and U937 cells, although larger less abundant transcripts are present. To study the origin of different fMLF-R transcripts, the genetic linkage of chemotactic receptor genes, and the regulation of fMLF-R gene expression, we determined the copy number, chromosomal location, structural organization, and 5'-flanking sequence of the human fMLF-R gene. BamHI restriction fragments derived from a human fMLF-R genomic cosmid clone were isolated, subcloned, and sequenced. These data indicate that the fMLF-R structural gene is approximately 7.5 kb in length and is comprised of two exons separated by an approximately 5.0-kb intron. The first exon encodes 66 bp of the 5'-untranslated sequence, while exon 2 encodes the coding and 3'-untranslated sequences. The genomic organization of the fMLF-R gene is similar to that of the adrenergic beta-1 and beta-2 G-protein-coupled receptor genes in that the coding sequence is contained in a single exon. The different 3'-untranslated sequences observed in fMLF-R cDNA clones are contiguous in the genomic structure, thereby indicating that these clones are derived in part by alternative polyadenylation. Southern blot analysis using human X hamster somatic cell hybrids and in situ hybridization indicated that the h-fMLF-R gene is located on chromosome 19q13.3. Primer extension experiments using dbcAMP-differentiated U937 RNA indicated a single transcriptional initiation site. Sequence analysis 5' of the transcriptional initiation site indicated possible cis-acting motifs that may regulate fMLF-R gene expression. These included AP-1 and CK-2 consensus sequences that bind nuclear factors of the Fos/Jun family and NF-GMb, respectively.