title: Determinants of thrombin receptor cleavage. Receptor domains involved, specificity, and role of the P3 aspartate.
abstract: Thrombin receptor cleavage at the Arg41- decreases -Ser42 peptide bond in the receptor's amino-terminal exodomain is necessary and sufficient for receptor activation. The rate of receptor cleavage at this site is a critical determinant of the magnitude of the cellular response to thrombin. These observations underscore the importance of defining the molecular basis for thrombin-receptor interaction and cleavage. We report that chimeric proteins bearing only thrombin receptor amino-terminal exodomain residues 36-60 are cleaved at rates similar to the wild-type thrombin receptor when expressed on the cell surface. A soluble amino-terminal exodomain protein was also cleaved efficiently by thrombin with a Km of 15-30 microM and k(cat) of approximately 50 s-1, with cleavage occurring only at the Arg41- decreases -Ser42 peptide bond. In the context of previous studies, these data suggest that the receptor's LDPR cleavage recognition sequence and DKYEPF hirudin-like domain account for thrombin-receptor interaction. Because a P3 aspartate in protein C's cleavage site inhibits cleavage by free thrombin, we investigated the role of the P3 aspartate in the receptor's LDPR sequence. Studies with mutant receptors revealed an inhibitory role for this residue only in the absence of the receptor's hirudin-like domain. These and other data suggest that the receptor's hirudin-like domain causes a conformational change in thombin's active center to accommodate the LDPR sequence and promote efficient receptor cleavage. Taken together, these studies imply that the thrombin receptor's amino-terminal exodomain contains all the machinery needed for efficient recognition and cleavage by thrombin. Thrombin appears to bind and cleave this domain independently of the rest of the receptor, with one thrombin molecule probably activating multiple receptors.
authors.affiliation: Cardiovascular Research Institute, University of California, San Francisco 94143, USA.
authors.forename: K
authors.initials: K
authors.lastname: Ishii
authors.forename: R
authors.initials: R
authors.lastname: Gerszten
authors.forename: Y W
authors.initials: YW
authors.lastname: Zheng
authors.forename: J B
authors.initials: JB
authors.lastname: Welsh
authors.forename: C W
authors.initials: CW
authors.lastname: Turck
authors.forename: S R
authors.initials: SR
authors.lastname: Coughlin
mesh_terms: D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism; D004252:DNA Mutational Analysis; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011485:Protein Binding; D018179:Receptors, Thrombin / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013917:Thrombin / Q000378:metabolism*
chemical_list: D018179:Receptors, Thrombin; D011993:Recombinant Fusion Proteins; D013917:Thrombin