Protease-Activated Receptor 4 Uses Anionic Residues To Interact with α-Thrombin in the Absence or Presence of Protease-Activated Receptor 1
Protease-Activated Receptor 4 Uses Anionic Residues To Interact with α-Thrombin in the Absence or Presence of Protease-Activated Receptor 1
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DOI：
10.1021/bi801334s
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发表时间：
2008-12-16
期刊：
影响因子：
2.9
通讯作者：
Nieman, Marvin T.
中科院分区：
文献类型：
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作者：
Nieman, Marvin T.
凝血酶激活蛋白酶激活受体1（PAR 1）的速度快于蛋白酶激活受体4（PAR 4），这是由于PART的胞外结构域中的水蛭素样序列与凝血酶的胞外位点I结合。然而，重组胞外结构域研究表明PAR 4确实与α-凝血酶具有延长的接触，这影响PAR 4的切割动力学。在本报告中，检查了PAR 4的胞外结构域中的阴离子簇（Asp（57）、Asp（59）、Glu（62）和Asp（65））在PAR 1不存在或存在的情况下对细胞上PAR 4的切割和活化的影响。α-凝血酶诱导野生型PAR 4（PAR 4-wt）钙流，EC（50）为110 nM，而四个阴离子残基（PAR 4-AAAA）的突变将EC（50）增加至641 nM。相比之下，PAR 4-wt和PAR 4-AAAA被具有相似EC（50）值（分别为588和449 nM; p = 0.48）的γ-凝血酶活化，表明α-凝血酶的外位点I在PAR 4活化中的作用。PAR 1的共表达使PAR 4-wt的切割EC（50）从321 nM降低到26 nM，PAR 4-AAAA的切割EC（50）从1.5 μ M降低到360 nM。在Asp（57）、Asp（59）、Glu（62）和Asp（65）处的单个点突变表明，PAR 4-D57 A被α-凝血酶激活，具有与PAR 4-wt（140 nM）相同的EC（50），而PAR 4-D59 A与PAR 4-AAAA（699 nM）相同。Glu（62）和Asp（65）有助于α-凝血酶识别，但程度较低。该报告表明，PAR 4使用其阴离子簇与α-凝血酶相互作用，并且即使在PAR 1存在下，这种相互作用也是重要的。
Thrombin activates protease-activated receptor 1 (PAR1) faster than protease-activated receptor 4 (PAR4) due to a hirudin-like sequence in the exodomain of PART that binds thrombin's exosite I. However, recombinant exodomain studies indicate that PAR4 does have extended contacts with a-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 mu M 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.