Protease-activated receptor-1 can mediate responses to SFLLRN in thrombin-desensitized cells: evidence for a novel mechanism for preventing or terminating signaling by PAR1's tethered ligand.
The thrombin receptor PAR1 is activated when thrombin cleaves the receptor's amino-terminal exodomain to reveal the new N-terminal sequence SFLLRN which then acts as a tethered peptide ligand. Free SFLLRN activates PAR1 independent of receptor cleavage and has been used to probe PAR1 function in various cells and tissues. PAR1-expressing cells desensitized to thrombin retain responsiveness to SFLLRN. Toward determining the mechanism of such responses, we utilized fibroblasts derived from a PAR1-deficient mouse. These cells were unresponsive to thrombin and SFLLRN and became sensitive to both ligands after transfection with human PAR1 cDNA. Moreover, PAR1-transfected cells responded to SFLLRN after thrombin-desensitization, indicating that signaling of thrombin-desensitized cells to SFLLRN was mediated by PAR1 itself. SFLLRN caused signaling in thrombin-desensitized cells when no uncleaved PAR1 was detectable on the cell surface; however, cleaved PAR1 was present. To determine whether the cleaved receptors could still signal, fibroblasts were transfected with a PAR1 mutant containing a trypsin site/SFLLRN sequence carboxyl terminal to the native thrombin site. These cells retained responsiveness to trypsin after thrombin-desensitization. Conversely, fibroblasts expressing a PAR1 mutant with the trypsin site/SFLLRN sequence amino terminal to the native thrombin site retained responsiveness to thrombin after trypsin-desensitization. This suggests that a population of thrombin-cleaved PAR1 can respond both to exogenous SFLLRN and to a second tethered ligand. In this population, the tethered ligand unmasked by thrombin cleavage must not be functional, suggesting the possibility of a novel mechanism of receptor shutoff involving sequestration or modification of the tethered ligand to prevent or terminate its function.
Cardiovascular Research Institute, Department of Medicine, Division of Endocrinology, University of California, San Francisco 94143, USA.
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Hammes
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Coughlin
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D005347:Fibroblasts / Q000201:enzymology / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D008024:Ligands; D008168:Lung / Q000166:cytology; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000494:pharmacology*; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D013917:Thrombin / Q000819:agonists / Q000494:pharmacology*; D013997:Time Factors
D008024:Ligands; D010446:Peptide Fragments; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; C082835:thrombin receptor peptide (42-47); D013917:Thrombin; D002118:Calcium