Biochemistry 2008, 47, 13279–13286
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Protease-Activated Receptor 4 Uses Anionic Residues To Interact with R-Thrombin in the Absence or Presence of Protease-Activated Receptor 1† Marvin T. Nieman* DiVision of Hematology/Oncology, Case Western ReserVe UniVersity School of Medicine, CleVeland, Ohio 44106-7284 ReceiVed July 15, 2008; ReVised Manuscript ReceiVed October 29, 2008
ABSTRACT:
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 R-thrombin that influence PAR4’s kinetics of cleavage. In this report, the role of an anionic cluster (Asp57, Asp59, Glu62, and Asp65) 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. R-Thrombin induces wild-type PAR4 (PAR4-wt) calcium flux with an EC50 of 110 nM, whereas mutation of the four anionic residues (PAR4-AAAA) increases the EC50 to 641 nM. In contrast, PAR4-wt and PAR4-AAAA are activated by γ-thrombin with similar EC50 values (588 and 449 nM, respectively; p ) 0.48), suggesting a role for R-thrombin’s exosite I in PAR4 activation. Coexpression of PAR1 lowered the EC50 of cleavage for PAR4-wt from 321 to 26 nM and for PAR4-AAAA from 1.5 µM to 360 nM. Individual point mutations at Asp57, Asp59, Glu62, and Asp65 show that PAR4-D57A is activated by R-thrombin with the same EC50 as PAR4-wt (140 nM) whereas PAR4-D59A is the same as PAR4-AAAA (699 nM). Glu62 and Asp65 contribute to R-thrombin recognition, but to a lesser extent. This report shows that PAR4 uses its anionic cluster to interact with R-thrombin and that this interaction is important even in the presence of PAR1.
Thrombin is the terminal enzyme of the clotting cascade. In addition to proteolyzing fibrinogen to fibrin, thrombin activates a variety of cell types via protease-activated receptors. The protease-activated receptor (PAR) family of G-protein-coupled receptors consists of four members (PAR1-4). PARs are activated by proteolysis of the Nterminal exodomain which exposes the tethered ligand (1). PAR1,1 -3, and -4 are primarily activated by thrombin, whereas PAR2 is activated by trypsin or tryptase (2). Human platelets express PAR1 and -4 (3). PAR1 is activated by low concentrations of thrombin, whereas PAR4 requires high concentrations. PAR4 is associated with a prolonged stimulus as measured by intracellular Ca2+ mobilization and may be required for stable clot formation (4, 5). Further, PAR4 cooperates with the P2Y12 receptor to mediate platelet aggregation (6). In contrast, PAR1-mediated platelet aggregation was independent of P2Y12 signaling. PAR1 signals through phosphoinositide 3-kinase (PI3K) and phosphatidic acid formed by phospholipase D (PLD) (7, 8). PAR4 signaling was not disrupted by inhibitors of PI3K or PLD. Therefore, these data suggest that PAR1 and PAR4 stimulate different signaling pathways in platelets and likely cooperate to mediate platelets’ complete response to thrombin. In † This research was supported by an American Heart Association Beginning Grant in Aid (0865441D to M.T.N.) and the flow cytometry core facility of the Comprehensive Cancer Center of Case Western Reserve University and University Hospitals Case Medical Center (P30 A43707). * To whom correspondence should be addressed. Telephone: (216) 368-4975. Fax: (216) 368-1166. E-mail:
[email protected]. 1 Abbreviations: PAR1, protease-activated receptor 1; PAR4, protease-activated receptor 4.
addition to platelet function, PAR4 participates in leukocyte rolling and may influence neutrophilic response in systemic inflammation contributing to multiorgan failure (9, 10). Collectively, PAR4 has physiological roles independent of that of PAR1. PAR1, like many thrombin substrates, interacts with thrombin’s exosite I for specificity and to lower the activation energy of cleavage at the active site (11-14). The exosite I interaction is mediated by a hirudin-like sequence in the PAR1 exodomain (15). In contrast, PAR4 does not have a hirudin-like sequence (16). On the basis of studies with peptides and recombinant exodomains, the primary sites of interaction of PAR4 with R-thrombin are at the thrombin cleavage site. In particular, amino acids Leu43 at P5, Pro44 at P4, and Pro46 at P2 are important R-thrombin interaction sites (17-19). However, individual point mutations at Leu43, Pro44, and Pro46 did not influence thrombin binding (i.e., did not influence the Km) but did reduce the rate of cleavage, indicating that Leu43, Pro44, and Pro46 are important for orienting PAR4 in the active site of thrombin for efficient cleavage (17). These data suggest that, like PAR1, PAR4 has extended contacts with R-thrombin that minimize the influence of the individual amino acids at the cleavage site (17). Earlier work by Jacques and Kuliopulos using purified exodomains demonstrates that mutations of the anionic cluster in the PAR4 exodomain (Asp57, Asp59, Glu62, and Asp65) increased the Km of R-thrombin binding 4-fold from 56 to 208 µM (18). Further experiments demonstrate that the anionic cluster stabilizes the interaction with R-thrombin by slowing the dissociation rate (18).
10.1021/bi801334s CCC: $40.75 2008 American Chemical Society Published on Web 11/20/2008
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Biochemistry, Vol. 47, No. 50, 2008
Nieman
Table 1: Amino Acid Sequences of the PAR4 Exodomain and Mutantsa sequence PAR4-wt PAR4-AAAA PAR4-D57A PAR4-S58A PAR4-D59A PAR4-T60A PAR4-E62A PAR4-D65A
T7-G18GT...P41SILPAPR*GYPGQVCANDSDTLELPDSSRALLLGWVPT... T7-G18GT...P41SILPAPR*GYPGQVCANASATLALPASSRALLLGWVPT... T7-G18GT...P41SILPAPR*GYPGQVCANASDTLELPDSSRALLLGWVPT... T7-G18GT...P41SILPAPR*GYPGQVCANDADTLELPDSSRALLLGWVPT... T7-G18GT...P41SILPAPR*GYPGQVCANDSATLELPDSSRALLLGWVPT... T7-G18GT...P41SILPAPR*GYPGQVCANDSDALELPDSSRALLLGWVPT... T7-G18GT...P41SILPAPR*GYPGQVCANDSDTLALPDSSRALLLGWVPT... T7-G18GT...P41SILPAPR*GYPGQVCANDSDTLELPASSRALLLGWVPT...
a Sequences are shown with standard one-letter abbreviations. The T7 epitope (MASMTGGQQMG) was added to the amino terminus. The asterisks indicate the thrombin cleavage site. Point mutations are indicated as bold and underlined. For space reasons, amino acids 21-40 of PAR4 are not shown.
The interaction of R-thrombin with PAR4 expressed on cells is less clear. PAR4 is cleaved with low efficiency when expressed on cells in the absence of PAR1 (18). However, in the presence of PAR1, as is the case on human platelets, PAR4 is activated more efficiently (20). PAR1 enhances PAR4 cleavage by remaining tightly bound to R-thrombin’s exosite I at PAR1’s hirudin-like sequence. The PAR1-bound R-thrombin cleaves an adjacent PAR4 molecule (20). A similar mechanism exists in mouse platelets with PAR3 and PAR4 (21). Therefore, it is unclear how PAR4 interacts with R-thrombin at exosite I in this model. Initial studies with human PAR4 demonstrate that R-thrombin and γ-thrombin activate PAR4 with the same efficiency (16). Since γ-thrombin is a proteolytic fragment of R-thrombin that does not have a functional exosite I (22), these data argue against PAR4 interacting with R-thrombin’s exosite I. However, the assessment of γ-thrombin cleaving human PAR4 was based on an end point assay that measured inositol phosphate production which may not reveal kinetic differences. This study indicates that the anionic cluster in the PAR4 exodomain (Asp57, Asp59, Glu62, and Asp65) contributes to PAR4’s recognition and cleavage by R-thrombin. In contrast, γ-thrombin cleaved wild-type PAR4 and PAR4 with mutations in the anionic cluster equally well. Further, the anionic cluster contributes to the interaction of PAR4 with R-thrombin in the presence of PAR1. Finally, the contribution of each of the residues in the anionic cluster was determined. These studies define an additional interaction site on PAR4 for R-thrombin in the presence of PAR1 and provide additional targets for the development of PAR activation antagonists. MATERIALS AND METHODS Antibodies and Reagents. Human R-thrombin (specific activity of 3593 NIH units/mg) and γ-thrombin (specific activity of
