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-formylmethionyl-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 [S-35]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 C beta(2) 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 C beta(2).