Cloning and expression analysis of a novel G-protein-coupled receptor selectively expressed on granulocytes

The migration of neutrophils into sites of acute and chronic inflammation i s mediated by chemokines. We used degenerate-primer reverse transcriptase-p olymerase chain reaction (RT-PCR) to analyze chemokine receptor expression in neutrophils and identify novel receptors. RNA was isolated from human pe ripheral blood neutrophils and front neutrophils that had been stimulated f or 5 h with granulocyte-macrophage colony-stimulating factor or by cocultur ing with primary human bronchial epithelial cells. Amplification products w ere cloned, and clone redundancy was determined. Seven known G-protein-coup led receptors were identified among 38 clones-CCR1, CCR4, CXCR1, CXCR2, CXC R4, HM63, and FPR1-as well as a novel gene, EX33, The full-length EX33 clon e was obtained, and an in silico approach was used to identify the putative murine homologue. The EX33 gene encodes a 396-amino-acid protein with limi ted sequence identity to known receptors, Expression studies of several kno wn chemokine receptors and EX33 revealed that resting neutrophils expressed higher levels of CXCRs and EX33 compared with activated neutrophils, North ern blot experiments revealed that EX33 is expressed mainly hi Bone marrow, lung, and peripheral blood leukocytes, Using RT-PCR analysis, we showed mo re abundant expression of EX33 in neutrophils and eosinophils, in compariso n with that in T- or B-lymphocytes, indicating cell-specific expression amo ng leukocytes.