Protease-activated receptor 4 uses anionic residues to interact with alpha-thrombin in the absence or presence of protease-activated receptor 1.
Thrombin activates protease-activated receptor 1 (PAR1) faster than protease-activated receptor 4 (PAR4) due to a hirudin-like sequence in the exodomain of PAR1 that binds thrombin's exosite I. However, recombinant exodomain studies indicate that PAR4 does have extended contacts with alpha-thrombin that influence PAR4's kinetics of cleavage. In this report, the role of an anionic cluster (Asp(57), Asp(59), Glu(62), and Asp(65)) in the exodomain of PAR4 is examined for its influence on cleavage and activation of PAR4 on cells in the absence or presence of PAR1. Alpha-thrombin induces wild-type PAR4 (PAR4-wt) calcium flux with an EC(50) of 110 nM, whereas mutation of the four anionic residues (PAR4-AAAA) increases the EC(50) to 641 nM. In contrast, PAR4-wt and PAR4-AAAA are activated by gamma-thrombin with similar EC(50) values (588 and 449 nM, respectively; p = 0.48), suggesting a role for alpha-thrombin's exosite I in PAR4 activation. Coexpression of PAR1 lowered the EC(50) of cleavage for PAR4-wt from 321 to 26 nM and for PAR4-AAAA from 1.5 microM to 360 nM. Individual point mutations at Asp(57), Asp(59), Glu(62), and Asp(65) show that PAR4-D57A is activated by alpha-thrombin with the same EC(50) as PAR4-wt (140 nM) whereas PAR4-D59A is the same as PAR4-AAAA (699 nM). Glu(62) and Asp(65) contribute to alpha-thrombin recognition, but to a lesser extent. This report shows that PAR4 uses its anionic cluster to interact with alpha-thrombin and that this interaction is important even in the presence of PAR1.
Nieman
Marvin T
MT
Division of Hematology/Oncology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-7284, USA. nieman@case.edu
D000595:Amino Acid Sequence; D000838:Anions; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D044463:Receptor, PAR-1 / Q000235:genetics / Q000378:metabolism*; D018179:Receptors, Thrombin / Q000235:genetics / Q000378:metabolism*; D013917:Thrombin / Q000235:genetics / Q000378:metabolism*
D000838:Anions; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; D013917:Thrombin; C112908:protease-activated receptor 4