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.
Hammes
S R
SR
Cardiovascular Research Institute, Department of Medicine, Division of Endocrinology, University of California, San Francisco 94143, USA.
Coughlin
S R
SR
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