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.
Müller
E
E
Department of Pediatrics, Faculty of Medicine, Université de Sherbrooke, Canada.
Dagenais
P
P
Alami
N
N
Rola-Pleszczynski
M
M
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