The long range goal of this project is to understand the mechanisms by which human blood-derived phagocytic cells localize to and become activated at sites of microbial invasion of the host. The first step toward the achievement of this goal was to establish the molecular structure and functional properties of chemoattractant receptors for human phagocytic cells. To this end, we have cloned cDNA,; encoding the N-formyl peptide receptor (FPR) and a low affinity interleukin-8 receptor (IL-8R) from an HL60 neutrophil cDNA library. The functional identity of cach receptor was proven by the acquisition of ligand-specific calcium mobilization in Xenopus oocytes that had been microinjected with clone-specific cRNA. A human complementary factor(s) encoded by 3.5 kb transcript(s) was discovered that is required for functional FPR expression but not for lL-8R expression in the oocyte. A third cDNA, designated FPRL1, was isolated that encodes a putative receptor that possesses 69% amino acid identity to FPR but that is not a calcium-mobilizing N-formyl peptide receptor. The lL-8R can be cross-activated by two related cytokines, gro and NAP-2. The low affinity lL-8R is 70% identical to a high affinity rabbit lL-8R. Comparison of the two sequences suggests strongly that the binding site for ligand resides in the amino terminal segment of each receptor. FPR and FPRL1 genes are on human chromosome 19 whereas the IL-8R gene is on human chromosome 2. Sequence comparisons identified all four receptors as members of a peptide chemoattractant receptor gene family. The third cytoplasmic loop of all four receptors as well as the C5a receptor is predicted to assume the secondary structure of a cationic amphipathic alpha helix which likely interacts with a common pertussis toxin sensitive G protein. In the course of our oocyte expression studies, we discovered a novel calcium transporter that is activated by the guanine nucleotide binding protein Gs and is potentiated by lanthanum ion over a narrow concentration range.
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