The carboxyl tail of protease-activated receptor-1 is required for chemotaxis. Correlation of signal termination and directional migration.
The G protein-coupled thrombin receptor, protease-activated receptor 1 (PAR1), mediates many of the actions of thrombin on cells including chemotaxis. In contrast to the reversible agonist binding that regulates signaling by most G protein-coupled receptors (GPCRs), PAR1 is activated by an irreversible proteolytic mechanism. Although activated PAR1 is phosphorylated, uncoupled, and internalized like typical GPCRs, signal termination is additionally dependent on lysosomal degradation of cleaved and activated receptors. In the present study we exploit two PAR1 mutants to examine the link between chemotaxis and receptor shutoff. One, a carboxyl tail deletion mutant (Y397Z), is defective in phosphorylation and internalization. The other, a carboxyl tail chimeric receptor (P/S), is phosphorylated and internalized upon activation but recycles to the plasma membrane like reversibly activated GPCRs. Expression of these receptors in a hematopoietic cell line disrupted cell migration along thrombin gradients. Thrombin activation of cells expressing P/S or Y397Z resulted in persistent signaling independent of the continued presence of thrombin. Signaling in response to the soluble agonist peptide SFLLRN was reversible for P/S but persisted for Y397Z. Strikingly, cells expressing P/S responded chemokinetically to thrombin but chemotactically to SFLLRN. In contrast, Y397Z-mediated migration was largely chemokinetic to both agonists. These studies suggest that termination of PAR1 signaling at the level of the receptor is necessary for gradient detection and directional migration.
Sambrano
G R
GR
Cardiovascular Research Institute, University of California, San Francisco, California 94143-0130, USA.
Coughlin
S R
SR
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002633:Chemotaxis / Q000502:physiology*; D006801:Humans; D051379:Mice; D009154:Mutation; D010446:Peptide Fragments / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000378:metabolism; D010766:Phosphorylation; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D013917:Thrombin / Q000494:pharmacology
D010446:Peptide Fragments; D010716:Phosphatidylinositols; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; C082835:thrombin receptor peptide (42-47); D013917:Thrombin; D002118:Calcium