How the protease thrombin talks to cells

Abstract

How does a protease act like a hormone to regulate cellular functions? The coagulation protease thrombin (EC 3.4.21.5) activates platelets and regulates the behavior of other cells by means of G protein-coupled protease-activated receptors (PARs). PAR1 is activated when thrombin binds to and cleaves its amino-terminal exodomain to unmask a new receptor amino terminus. This new amino terminus then serves as a tethered peptide ligand, binding intramolecularly to the body of the receptor to effect transmembrane signaling. The irreversibility of PAR1's proteolytic activation mechanism stands in contrast to the reversible ligand binding that activates classical G protein-coupled receptors and compels special mechanisms for desensitization and resensitization. In endothelial cells and fibroblasts, activated PAR1 rapidly internalizes and then sorts to lysosomes rather than recycling to the plasma membrane as do classical G protein-coupled receptors. This trafficking behavior is critical for termination of thrombin signaling. An intracellular pool of thrombin receptors refreshes the cell surface with naïve receptors, thereby maintaining thrombin responsiveness. Thus cells have evolved a trafficking solution to the signaling problem presented by PARs. Four PARs have now been identified. PAR1, PAR3, and PAR4 can all be activated by thrombin. PAR2 is activated by trypsin and by trypsin-like proteases but not by thrombin. Recent studies with knockout mice, receptor-activating peptides, and blocking antibodies are beginning to define the role of these receptors in vivo.

Figure 1

Mechanism of PAR1 activation. Thrombin (large sphere) recognizes the amino-terminal exodomain of the G protein-coupled thrombin receptor PAR1. This interaction utilizes sites both amino-terminal (P1–P4, small sphere) and carboxyl-terminal (P9′–P14′, small oval) to the thrombin cleavage site. Thrombin cleaves the peptide bond between receptor residues Arg-41 and Ser-42. This serves to unmask a new amino terminus beginning with the sequence SFLLRN (diamond) that functions as a tethered ligand, docking intramolecularly with the body of the receptor to effect transmembrane signaling. hPAR1, human PAR1; the asterisk indicates the activated form. Synthetic SFLLRN peptide will function as an agonist, bypassing the requirement for receptor cleavage.

Figure 2

Protease-activated receptor family. Four PARs are known. Amino acid sequence identity between human (h-) and mouse (m-) homologues of each is approximately 60%, but identity between different PARs within a single species falls to approximately 30%. Xen indicates Xenopus. Human PAR1, PAR3, and PAR4 can be activated by thrombin, and sensing thrombin is likely, at least in part, their role in vivo (see text). One receptor, PAR2, is activated by trypsin and tryptase but not by thrombin. Its roles in vivo remain to be explored. The four PAR genes share a common two-exon structure. In essence, the first exon encodes a signal peptide and the second the mature receptor protein. The genes encoding PARs 1, 2, and 3 are adjacent in the mouse and human genomes, whereas the PAR4 gene resides at a separate location (32, 65, 66).