Thrombin receptor activation. Confirmation of the intramolecular tethered liganding hypothesis and discovery of an alternative intermolecular liganding mode.

Abstract

Cleavage of the thrombin receptor's amino-terminal exodomain at the Arg41/Ser42 peptide bond within the sequence ... LDPR41/S42FLLRN ... is necessary and sufficient for receptor activation by proteases. The synthetic peptide SFLLRN activates the receptor independent of proteolysis. We proposed that the SFLLRN sequence is a tethered peptide ligand; receptor cleavage unmasks this agonist which then binds intramolecularly to effect receptor activation. The alternative hypothesis that receptor cleavage or exogenous SFLLRN effect receptor activation by disrupting tonic inhibitory interactions exerted by the receptor's amino-terminal exodomain has not been excluded. We report that delta AMINO, a mutant thrombin receptor lacking the amino-terminal exodomain, was not constitutively active and responded to SFLLRN but not thrombin when expressed in Xenopus oocytes or mammalian cells. Thrombin signaling was restored when delta AMINO was co-expressed with ATE-CD8 which encoded the receptor's amino-terminal exodomain fused to the transmembrane domain of CD8. Co-expression of a thrombin receptor lacking a functional tethered ligand domain ("F43A") with a non-signaling receptor mutant bearing an intact tethered ligand domain ("YYY") also reconstituted thrombin signaling. However, the EC50 for thrombin activation of cells co-expressing F43A and YYY was > 1000-fold that for cells expressing comparable levels of wild type receptor, while EC50s for activation by SFLLRN were similar. These and other data refute the release from inhibition hypothesis and suggest that while intermolecular liganding between two thrombin receptor molecules can occur, the intramolecular tethered liganding mechanism is the predominant mode of thrombin receptor activation.