Molecular cloning of the fMet-Leu-Phe receptor from neutrophils.
The bacterial chemotactic peptide fMet-Leu-Phe (fMLP) activates neutrophils upon binding to surface receptors. In a previous communication we reported the functional reconstitution of the fMLP receptor in Xenopus laevis oocytes (Coats, W. D., and Navarro, J. (1990) J. Biol. Chem. 265, 5964-5966). In this work we report the isolation of the cDNA encoding the fMLP receptor from neutrophils. A rabbit neutrophil cDNA library was screened with an oligonucleotide probe deduced from the nucleotide sequence of G-protein-coupled receptors, and a cDNA encoding the fMLP receptor was isolated. This cDNA was characterized according to the following criteria: 1) Analysis of the deduced amino acid sequence revealed that the clone belongs to a G-protein-coupled receptor. 2) Tissue distribution analysis of the mRNA indicated that the message is only found in neutrophils. 3) In vitro translation of the message revealed a protein corresponding in size to the deglycosylated fMLP receptor. 4) X. laevis oocytes injected with the fMLP receptor message exhibited fMLP-dependent calcium mobilization and specific binding to the fMLP analog 125I-labeled fNle-Leu-Phe-Nle-Tyr-Lys (where Nle is norleucine and fNle is formylnorleucine). The molecular cloning of the fMLP receptor should provide the framework to analyze the relationship between structure, function, and regulation of this receptor.
Department of Physiology, Boston University School of Medicine, Massachusetts 02118.
K M
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Thomas
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Pyun
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Navarro
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D005260:Female; D015870:Gene Expression; D015723:Gene Library; D008969:Molecular Sequence Data; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000378:metabolism*; D009504:Neutrophils / Q000378:metabolism*; D015345:Oligonucleotide Probes; D009865:Oocytes / Q000378:metabolism; D014176:Protein Biosynthesis; D012313:RNA / Q000097:blood / Q000235:genetics / Q000302:isolation & purification; D011817:Rabbits; D012689:Sequence Homology, Nucleic Acid; D014982:Xenopus laevis
D015345:Oligonucleotide Probes; D009240:N-Formylmethionine Leucyl-Phenylalanine; D012313:RNA; D004247:DNA