Cloning and characterization of human protease-activated receptor 4

Abstract

Protease-activated receptors 1-3 (PAR1, PAR2, and PAR3) are members of a unique G protein-coupled receptor family. They are characterized by a tethered peptide ligand at the extracellular amino terminus that is generated by minor proteolysis. A partial cDNA sequence of a fourth member of this family (PAR4) was identified in an expressed sequence tag database, and the full-length cDNA clone has been isolated from a lymphoma Daudi cell cDNA library. The ORF codes for a seven transmembrane domain protein of 385 amino acids with 33% amino acid sequence identity with PAR1, PAR2, and PAR3. A putative protease cleavage site (Arg-47͞Gly-48) was identified within the extracellular amino terminus. COS cells transiently transfected with PAR4 resulted in the formation of intracellular inositol triphosphate when treated with either thrombin or trypsin. A PAR4 mutant in which the Arg-47 was replaced with Ala did not respond to thrombin or trypsin. A hexapeptide (GYPGQV) representing the newly exposed tethered ligand from the amino terminus of PAR4 after proteolysis by thrombin activated COS cells transfected with either wild-type or the mutant PAR4. Northern blot showed that PAR4 mRNA was expressed in a number of human tissues, with high levels being present in lung, pancreas, thyroid, testis, and small intestine. By f luorescence in situ hybridization, the human PAR4 gene was mapped to chromosome 19p12.

Platelet PAR1 receptor density—Correlation to platelet activation response and changes in exposure after platelet activation

“…Both are coupled to Gαq and Gα 12/13 [1]. Thrombin cleaves the receptors within the large N-terminal extracellular domain, creating new amino terminals, SFLLRN and GYPGQV respectively [2,3]. The new N-terminus formed by thrombin cleavage serves as a "tethered ligand" [4] which binds intramolecularly and causes receptor activation.…”

Mechanism of trypsin‐induced endothelium‐dependent vasorelaxation in the porcine coronary artery

“…The activation of PAR is initiated by the proteolytic cleavage of its extracellular domain to create the new N-terminus that functions as a tethered ligand (Dery et al, 1998). Four members of PAR, PAR-1, PAR-2, PAR-3 and PAR-4, have so far been isolated and cloned (Ishihara et al, 1997;Kahn et al, 1998;Nystedt et al, 1994;Vu et al, 1991;Xu et al, 1998). PAR-1, PAR-3 and PAR-4 serve as the receptor for thrombin, while PAR-2 and PAR-4 serve as the receptor for trypsin (Nystedt et al, 1994;Xu et al, 1998).…”

“…Four members of PAR, PAR-1, PAR-2, PAR-3 and PAR-4, have so far been isolated and cloned (Ishihara et al, 1997;Kahn et al, 1998;Nystedt et al, 1994;Vu et al, 1991;Xu et al, 1998). PAR-1, PAR-3 and PAR-4 serve as the receptor for thrombin, while PAR-2 and PAR-4 serve as the receptor for trypsin (Nystedt et al, 1994;Xu et al, 1998). The synthetic peptides corresponding to the tethered ligand regions have been shown to activate PARs without cleavage, and have also been successfully used as a useful tool to investigate the functional role of PARs in the cellular e ects of thrombin and trypsin (Dery et al, 1998).…”

Thrombin downregulates muscle acetylcholine receptors via an IP3 signaling pathway by activating its G‐protein‐coupled protease‐activated receptor‐1

“…ß 2003 An emerging family of cell-surface protease-activated receptors (PARs) has been localized on various cellular types such as neurons, glial cells, fibroblasts (Déry et al, 1998), and skeletal muscle cells . PAR-1, PAR-3, and PAR-4 (Vu et al, 1991;Ishihara et al, 1997;Xu et al, 1998) are coupled to G-proteins and their activation by the proteolytic action of thrombin transduces intracellular signals. The serine protease thrombin, first described as cleaving fibrinogen into fibrin in blood clotting, also regulates multiple extravascular cellular processes by activating its receptor PAR-1 (Grand et al, 1996).…”