THROMBIN PRIMES RESPONSIVENESS OF SELECTIVE CHEMOATTRACTANT RECEPTORSAT A SITE DISTAL TO G-PROTEIN ACTIVATION

To define the molecular basis of human chemoattractant receptor regula tion, rat basophilic leukemia RBL-2H3 cells, which are thrombin-respon sive, were transfected to stably express epitope-tagged receptors for C5a, interleukin-8 (IL-8), formylpeptides (e.g. N-formylmethionyl-leuc yl phenylalanine (fMLP)), and platelet-activating factor (PAF), Here w e demonstrate that both thrombin and a synthetic peptide ligand for th e thrombin receptor (sequence SFLLRN) caused phosphorylation and heter ologous desensitization of the receptors for C5a, IL-8, and PAF but no t that for formylpeptides as measured by agonist stimulated [S-35]guan osine 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, an d degranulation stimulated by PAF, Unexpectedly, despite heterologous desensitization at the level of receptor/G protein activation, there w as enhancement (''priming'') by thrombin of subsequent activities stim ulated 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 C a2+ mobilization in RBL-2H3 cells but did not cause priming, In additi on, SFLLRN and the thrombin receptor antagonist peptide FLLRN both inh ibited 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+ mob ilization in RBL-2H3 cells, effectively primed the response to fMLP. T hese data demonstrate that heterologous receptor phosphorylation and a ttenuation of G protein activation are not, by themselves, sufficient for the inhibition of biological responses mediated by C5a and IL-8, M oreover, thrombin appears to utilize mechanism(s) independent of its t ethered 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 stri king selectivity of thrombin's priming may be mediated via its ability to enhance receptor-mediated activation of phospholipase C beta(2).