Thrombin is a coagulation protease that activates platelets, leukocytes, endothelial and mesenchymal cells at sites of vascular injury, acting partly through an unusual proteolytically activated G-protein-coupled receptor. Knockout of the gene encoding this receptor provided definitive evidence for a second thrombin receptor in mouse platelets and for tissue-specific roles for different thrombin receptors. We now report the cloning and characterization of a new human thrombin receptor, designated protease-activated receptor 3 (PAR3). PAR3 can mediate thrombin-triggered phosphoinositide hydrolysis and is expressed in a variety of tissues, including human bone marrow and mouse megakaryocytes, making it a candidate for the sought-after second platelet thrombin receptor. PAR3 provides a new tool for understanding thrombin signalling and a possible target for therapeutics designed selectively to block thrombotic, inflammatory and proliferative responses to thrombin.
The in vivo roles of the hundreds of mammalian G protein-coupled receptors (GPCRs) are incompletely understood. To explore these roles, we generated mice expressing the S1 subunit of pertussis toxin, a known inhibitor of G i/o signaling, under the control of the ROSA26 locus in a Cre recombinase-dependent manner (ROSA26 PTX ). Crossing ROSA26 PTX mice to mice expressing Cre in pancreatic β cells produced offspring with constitutive hyperinsulinemia, increased insulin secretion in response to glucose, and resistance to dietinduced hyperglycemia. This phenotype underscored the known importance of G i/o and hence of GPCRs for regulating insulin secretion. Accordingly, we quantified mRNA for each of the approximately 373 nonodorant GPCRs in mouse to identify receptors highly expressed in islets and examined the role of several. We report that 3-iodothyronamine, a thyroid hormone metabolite, could negatively and positively regulate insulin secretion via the G i -coupled α 2A -adrenergic receptor and the G s -coupled receptor Taar1, respectively, and proteaseactivated receptor-2 could negatively regulate insulin secretion and may contribute to physiological regulation of glucose metabolism. The ROSA26 PTX system used in this study represents a new genetic tool to achieve tissue-specific signaling pathway modulation in vivo that can be applied to investigate the role of G i/o -coupled GPCRs in multiple cell types and processes.
G protein-coupled receptors can trigger metalloproteinase-dependent shedding of proteins from the cell surface. We now report that G protein-coupled receptors can themselves undergo regulated metalloproteinasedependent shedding. The N-terminal exodomain of protease-activated receptor-1 (PAR1), a G protein-coupled receptor for thrombin, displayed regulated shedding in endothelial cells, which normally express this receptor. Cleavage occurred at a site predicted to render the receptor unresponsive to thrombin. A chimeric protein in which the N-terminal exodomain of PAR1 was fused to an unrelated transmembrane segment was shed as efficiently as PAR1, shedding of both proteins was stimulated by phorbol ester and by a PAR1 agonist. TNF␣ protease inhibitor-2 (TAPI-2), phenanthroline, and tissue inhibitor of metalloproteinase-3 (TIMP-3) but not TIMP-1 or -2 inhibited such shedding. These and other data suggest that the information that specifies PAR1 shedding resides within its N-terminal exodomain rather than its heptahelical segment, that activation of protein kinase C or of PAR1 itself can stimulate PAR1 shedding in trans, and that ADAM17/TACE or a metalloproteinase with similar properties mediates PAR1 shedding. Regulated shedding reduced the amount of cell surface PAR1 available for productive cleavage by thrombin by half or more, but thus far we have been unable to demonstrate an effect of PAR1 shedding on cellular responsiveness to thrombin. Nonetheless, regulated shedding of G protein-coupled receptors represents a new mechanism by which signaling by this important class of receptors might be modulated.
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.
Southern blot analysis of mouse genomic DNA reveals two EcoRI fragments which faintly hybridize to mouse Adh-1 cDNA and are not part of the Adh-1 gene. These fragments were isolated from agarose gels, cloned, and characterized. Sequence analysis of the 2.1-kb EcoRI fragment suggests that it is likely a pseudogene since it does not contain a long open reading frame. However; the 2.0-kb EcoRI fragment contains a coding sequence with a long open reading frame which corresponds to exon 6 of the mouse Adh-1 gene. Comparison of the coding sequence with other known ADHs suggests that the sequence has diverged sufficiently from any currently known class of ADH to be a possible distinct class. Further confirmation awaits analysis of currently available genomic clones. Using these sequences as probe, restriction fragment length polymorphisms were identified for each sequence between C57Bl/6J and DBA/2J inbred mouse strains. The strain distribution pattern for these allelic differences was determined among the B x D recombinant inbred strains. This analysis revealed that the 2.1-kb EcoRI sequence is located on chromosome 3 but at a distance from the Adh-1/Adh-3 complex as previously reported. However, the new polymorphism identified in the 2.0-kb EcoRI fragment enabled this sequence to be mapped at the Adh-1/Adh-3 complex.
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