Thrombin primes responsiveness of selective chemoattractant receptors at a site distal to G protein activation.
To define the molecular basis of human chemoattractant receptor regulation, rat basophilic leukemia RBL-2H3 cells, which are thrombin-responsive, were transfected to stably express epitope-tagged receptors for C5a, interleukin-8 (IL-8), formylpeptides (e.g. N-formyl-methionyl-leucyl-phenylalanine (fMLP)), and platelet-activating factor (PAF). Here we demonstrate that both thrombin and a synthetic peptide ligand for the thrombin receptor (sequence SFLLRN) caused phosphorylation and heterologous desensitization of the receptors for C5a, IL-8, and PAF but not that for formylpeptides as measured by agonist-stimulated [35S]guanosine 5'-3-O-(thio)triphosphate binding to membranes. Consistent with the PAF receptor phosphorylation, both thrombin and thrombin receptor peptide inhibited phosphoinositide hydrolysis, Ca2+ mobilization, and degranulation stimulated by PAF. Unexpectedly, despite heterologous desensitization at the level of receptor/G protein activation, there was enhancement ("priming") by thrombin of subsequent activities stimulated by C5a and IL-8 as well as fMLP. The priming effect of thrombin was blocked by its inhibitor, hirudin. However, two other activators of the thrombin receptor, the peptide SFLLRN and trypsin, stimulated Ca2+ mobilization in RBL-2H3 cells but did not cause priming. In addition, SFLLRN and the thrombin receptor antagonist peptide FLLRN both inhibited thrombin-induced Ca2+ mobilization but not priming. Furthermore, the proteolytically active gamma-thrombin, which does not stimulate the tethered ligand thrombin receptor and caused little or no Ca2+ mobilization in RBL-2H3 cells, effectively primed the response to fMLP. These data demonstrate that heterologous receptor phosphorylation and attenuation of G protein activation are not, by themselves, sufficient for the inhibition of biological responses mediated by C5a and IL-8. Moreover, thrombin appears to utilize mechanism(s) independent of its tethered ligand receptor to selectively prime phospholipase C-mediated biological responses of the C5a, IL-8, and formylpeptide receptors but not PAF. Because C5a, IL-8, and formylpeptide activate phospholipase Cbeta2, whereas PAF stimulates a different phospholipase C, the striking selectivity of thrombin's priming may be mediated via its ability to enhance receptor-mediated activation of phospholipase Cbeta2.
Ali
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Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
Tomhave
E D
ED
Richardson
R M
RM
Haribabu
B
B
Snyderman
R
R
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis / Q000302:isolation & purification / Q000502:physiology*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D015936:Complement C5a / Q000494:pharmacology; D010450:Endopeptidases / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007294:Inositol / Q000378:metabolism; D007295:Inositol Phosphates / Q000378:metabolism; D016209:Interleukin-8 / Q000494:pharmacology; D007700:Kinetics; D015471:Leukemia, Basophilic, Acute; D008969:Molecular Sequence Data; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D010766:Phosphorylation; D010980:Platelet Membrane Glycoproteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000502:physiology*; D051381:Rats; D044087:Receptor, Anaphylatoxin C5a; D011956:Receptors, Cell Surface; D011951:Receptors, Complement / Q000096:biosynthesis / Q000302:isolation & purification / Q000502:physiology*; D044042:Receptors, Formyl Peptide; D043562:Receptors, G-Protein-Coupled; D011971:Receptors, Immunologic / Q000096:biosynthesis / Q000302:isolation & purification / Q000502:physiology*; D018123:Receptors, Interleukin / Q000096:biosynthesis / Q000302:isolation & purification / Q000502:physiology*; D023062:Receptors, Interleukin-8A; D018000:Receptors, Peptide / Q000096:biosynthesis / Q000302:isolation & purification / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D016324:Sequence Tagged Sites; D013917:Thrombin / Q000494:pharmacology*; D014162:Transfection; D014407:Tumor Cells, Cultured; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
D015703:Antigens, CD; D007295:Inositol Phosphates; D016209:Interleukin-8; D010446:Peptide Fragments; D010980:Platelet Membrane Glycoproteins; D044087:Receptor, Anaphylatoxin C5a; D011956:Receptors, Cell Surface; D011951:Receptors, Complement; D044042:Receptors, Formyl Peptide; D043562:Receptors, G-Protein-Coupled; D011971:Receptors, Immunologic; D018123:Receptors, Interleukin; D023062:Receptors, Interleukin-8A; D018000:Receptors, Peptide; D011994:Recombinant Proteins; C039351:platelet activating factor receptor; C082835:thrombin receptor peptide (42-47); D007294:Inositol; D009240:N-Formylmethionine Leucyl-Phenylalanine; D015936:Complement C5a; D001619:beta-N-Acetylhexosaminidases; D010450:Endopeptidases; D013917:Thrombin; D019204:GTP-Binding Proteins; D002118:Calcium