Nieman Marvin T
Division of Hematology/Oncology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-7284, USA.
Biochemistry. 2008 Dec 16;47(50):13279-86. doi: 10.1021/bi801334s.
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.
由于蛋白酶激活受体1（PAR1）胞外域中存在一个与凝血酶外位点I结合的水蛭素样序列，凝血酶激活PAR1的速度比蛋白酶激活受体4（PAR4）快。然而，重组胞外域研究表明，PAR4确实与α-凝血酶有广泛的接触，这影响了PAR4的裂解动力学。在本报告中，研究了PAR4胞外域中一个阴离子簇（Asp(57)、Asp(59)、Glu(62)和Asp(65)）在不存在或存在PAR1的情况下对PAR4在细胞上的裂解和激活的影响。α-凝血酶诱导野生型PAR4（PAR4-wt）钙通量，EC(50)为110 nM，而四个阴离子残基的突变（PAR4-AAAA）使EC(50)增加到641 nM。相比之下，PAR4-wt和PAR4-AAAA被γ-凝血酶激活，EC(50)值相似（分别为588和449 nM；p = 0.48），表明α-凝血酶的外位点I在PAR4激活中起作用。PAR1的共表达将PAR4-wt的裂解EC(50)从321 nM降低到26 nM，将PAR4-AAAA的裂解EC(50)从1.5 μM降低到360 nM。在Asp(57)、Asp(59)、Glu(62)和Asp(65)处的单个点突变表明，PAR4-D57A被α-凝血酶激活的EC(50)与PAR4-wt相同（140 nM），而PAR4-D59A与PAR4-AAAA相同（699 nM）。Glu(62)和Asp(65)有助于α-凝血酶的识别，但程度较小。本报告表明，PAR4利用其阴离子簇与α-凝血酶相互作用，并且即使在存在PAR
