The human leukocyte platelet-activating factor receptor. cDNA cloning, cell surface expression, and construction of a novel epitope-bearing analog.
A human myeloid transcript of approximately 4 kilobases was cloned as a cDNA from an expression library based on homology with the guinea pig cDNA recently described by Honda et al. (Honda, Z-I., Nakamura, M., Miki, I., Minami, M., Watanabe, T., Seyama, Y., Okado, H., Tok, H., Ito, K., Miyamato, T., and Shimizu, T. (1991) Nature 349, 342-346) as a receptor for platelet-activating factor (PAF). The cloned DNA confers high affinity binding sites for platelet-activating factor when transfected into COS-7 cells and has binding and desensitization properties similar to the human leukocyte receptor. Southern analysis using this cDNA indicates that the PAF receptor gene is present as a single copy in the human genome. The deduced protein sequence predicts seven hydrophobic regions for the PAF receptor, characteristic of the rhodopsin gene family, and is 83% identical to the deduced protein sequence of the corresponding guinea pig molecule. A modified human PAF receptor cDNA was constructed by inserting an additional 30 nucleotides after the 5'-ATG, encoding the amino acid sequence MDYKDDDDKEF, which is specifically recognized by a monoclonal antibody. The modified cDNA encodes a functional PAF receptor and is detected by antibody on the membrane of transfected COS-7 cells. The use of this construct supports the structural model for the rhodopsin-like superfamily of receptors which places the NH2-terminal sequence on the extracellular side of the membrane, and should additionally be useful for affinity purification of the receptor protein.
Kunz
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Department of Pediatrics, Children's Hospital, Boston, Massachusetts 02115.
Gerard
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Gerard
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D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D000939:Epitopes / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D007150:Immunohistochemistry; D007962:Leukocytes / Q000378:metabolism*; D008168:Lung / Q000378:metabolism; D008969:Molecular Sequence Data; D010972:Platelet Activating Factor / Q000378:metabolism*; D010980:Platelet Membrane Glycoproteins; D011487:Protein Conformation; D012313:RNA / Q000235:genetics / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000276:immunology; D043562:Receptors, G-Protein-Coupled; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010972:Platelet Activating Factor; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; C039351:platelet activating factor receptor; D012313:RNA; D004247:DNA