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

Abstract

The migration of neutrophils into sites of acute and chronicinflammation is mediated by chemokines. We used degenerate-primerreverse transcriptase-polymerase chain reaction (RT-PCR) to analyzechemokine receptor expression in neutrophils and identify novelreceptors. RNA was isolated from human peripheral blood neutrophils andfrom neutrophils that had been stimulated for 5 h withgranulocyte-macrophage colony-stimulating factor or by coculturing withprimary human bronchial epithelial cells. Amplification products werecloned, and clone redundancy was determined. Seven knownG-protein-coupled receptors were identified among 38 clones—CCR1,CCR4, CXCR1, CXCR2, CXCR4, HM63, and FPR1—as well as a novel gene,EX33. The full-length EX33 clone was obtained, and an insilico approach was used to identify the putative murine homologue. TheEX33 gene encodes a 396-amino-acid protein with limitedsequence identity to known receptors. Expression studies of severalknown chemokine receptors and EX33 revealed that resting neutrophilsexpressed higher levels of CXCRs and EX33 compared with activatedneutrophils. Northern blot experiments revealed that EX33 is expressedmainly in bone marrow, lung, and peripheral blood leukocytes. UsingRT-PCR analysis, we showed more abundant expression of EX33 inneutrophils and eosinophils, in comparison with that in T- or B-lymphocytes, indicating cell-specific expression amongleukocytes.