Identification and functional characterization of platelet-activating factor receptors in human leukocyte populations using polyclonal anti-peptide antibody.
Recently, the successful cloning of a receptor for platelet-activating factor (PAF), a lipid mediator of inflammation, was reported. Here we investigated the distribution and potential diversity of human PAF receptors (hPAF-Rs) among individual leukocyte populations by (i) hPAF-R mRNA transcription studies and (ii) analysis of cell surface expression of hPAF-R protein using a polyclonal anti-peptide antibody (anti-hPAF-R164-173). Northern blot analysis, flow cytometry, and immunoblotting with anti-hPAF-R antibody indicated that monocytic, neutrophilic, and B-lymphocytic cell lines all shared a similar hPAF-R species, whereas resting T-cell and natural killer cell lines failed to express detectable levels of either hPAF-R protein or mRNA. Peripheral blood leukocyte populations showed a distribution of hPAF-R cell surface expression similar to that of the corresponding cell lines. Furthermore, binding of anti-hPAF-R164-173 antiserum, purified IgG, or Fab and F(ab')2 fragments to the receptor of all investigated PAF-R-positive cell lines induced an increase in intracellular free calcium concentration. The characterization of the expression of a lipid ligand receptor using antibodies against an intrinsic portion of the receptor protein has, to our knowledge, never been reported previously.
Department of Pediatrics, Faculty of Medicine, Université de Sherbrooke, Canada.
E
E
Müller
P
P
Dagenais
N
N
Alami
M
M
Rola-Pleszczynski
D015152:Blotting, Northern; D002118:Calcium / Q000378:metabolism; D004247:DNA; D005434:Flow Cytometry; D015870:Gene Expression; D006801:Humans; D007962:Leukocytes / Q000378:metabolism*; D010972:Platelet Activating Factor / Q000378:metabolism / Q000494:pharmacology*; D010980:Platelet Membrane Glycoproteins; D012333:RNA, Messenger / Q000302:isolation & purification / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D010972:Platelet Activating Factor; D010980:Platelet Membrane Glycoproteins; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; C039351:platelet activating factor receptor; D004247:DNA; D002118:Calcium