Differential cholera-toxin- and pertussis-toxin-catalysed ADP-ribosylation of G-proteins coupled to formyl-peptide and leukotriene B4 receptors.
N-Formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe) and leukotriene B4 (LTB4) induce disparate second-messenger generation and functional responses in neutrophils and HL-60 granulocytes. Receptors for these chemoattractants couple to a common pool of G-proteins which are substrates for both pertussis-toxin- and cholera-toxin-catalysed ADP-ribosylation. The hypothesis that formyl-peptide and LTB4 receptors induce different receptor-specific conformations of activated G-proteins was tested. The ability of pertussis toxin and cholera toxin to ADP-ribosylate G(i) proteins coupled to formyl-peptide or LTB4 receptors in membranes isolated from HL-60 granulocytes was used to assess the conformational state of the alpha subunits. Cholera-toxin-catalysed ADP-ribosylation of alpha 40 (40 kDa alpha subunit) was inhibited by guanosine 5'-[beta gamma-imido]triphosphate and GDP in a concentration-dependent manner. Addition of fMet-Leu-Phe, but not LTB4, re-established cholera-toxin labelling of alpha 40 in the presence of either guanine nucleotide. In the absence of guanine nucleotides, fMet-Leu-Phe and C5a enhanced cholera-toxin-catalysed labelling of alpha 40, whereas LTB4 and platelet-activating factor had no effect. Preincubation with fMet-Leu-Phe, but not LTB4, inhibited pertussis-toxin labelling of alpha 40 in the presence of guanosine 5'-[gamma-thio]triphosphate and in the absence of guanine nucleotides. Preincubation with fMet-Leu-Phe or LTB4 enhanced pertussis-toxin labelling of alpha 40 in the presence of GDP. These data suggest that activated G(i) proteins coupled to formyl-peptide and LTB4 receptors exist in different conformations determined by the receptor with which they interact.
Department of Medicine, University of Louisville Health Sciences Center, KY.
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Schepers
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McLeish
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D002772:Cholera Toxin / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute; D007975:Leukotriene B4 / Q000494:pharmacology*; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D009243:NAD / Q000378:metabolism*; D037342:Pertussis Toxin; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000187:drug effects / Q000378:metabolism*; D018102:Receptors, Leukotriene B4; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D018102:Receptors, Leukotriene B4; D010566:Virulence Factors, Bordetella; D009243:NAD; D006153:Guanosine Diphosphate; D007975:Leukotriene B4; D000246:Adenosine Diphosphate Ribose; D006165:Guanylyl Imidodiphosphate; D009240:N-Formylmethionine Leucyl-Phenylalanine; D002772:Cholera Toxin; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins