Mapping Human Protease-activated Receptor 4 (PAR4) Homodimer Interface to Transmembrane Helix 4
Mapping Human Protease-activated Receptor 4 (PAR4) Homodimer Interface to Transmembrane Helix 4
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DOI：
10.1074/jbc.m112.341438
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发表时间：
2012-03-23
影响因子：
4.8
通讯作者：
Nieman, Marvin T.
中科院分区：
文献类型：
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作者：
de la Fuente, Maria;Noble, Daniel N.;Nieman, Marvin T.
凝血酶通过结合和切割蛋白酶激活受体1和4（PAR 1和PAR 4）激活血小板。由于PAR 4活化对人类和小鼠血小板的重要性以及PAR 4在其他组织中的新兴作用，进行了实验以表征PAR 4同源二聚体之间的相互作用。双分子荧光互补和生物发光共振能量转移（BRET）被用来检查PAR 4同源二聚体界面。在双分子荧光互补实验中，PAR 4形成的同源二聚体被破坏的未标记的PAR 4在浓度依赖性的方式，但不是由视紫红质。在BRET实验中，PAR 4同源二聚体显示出特异性相互作用，如响应于增加的PAR 4-GFP表达的双曲线BRET信号所指示的。PAR 4在BRET测定中不与视紫红质相互作用。未标记的PAR 4以浓度依赖性方式破坏阈值最大BRET信号。相反，视紫红质不能破坏BRET信号，表明PAR 4同源二聚体的破坏不是由于非特异性相互作用。一组rho-PAR 4嵌合体和PAR 4点突变体已经将二聚体界面映射到跨膜螺旋4中的疏水残基。最后，破坏二聚体形成的突变降低了响应于PAR 4激动剂肽的钙动员。这些结果将二聚体形成的损失与PAR 4信号传导的损失联系起来。
Thrombin activates platelets by binding and cleaving protease-activated receptors 1 and 4 (PAR1 and PAR4). Because of the importance of PAR4 activation on platelets in humans and mice and emerging roles for PAR4 in other tissues, experiments were done to characterize the interaction between PAR4 homodimers. Bimolecular fluorescence complementation and bioluminescence resonance energy transfer (BRET) were used to examine the PAR4 homodimer interface. In bimolecular fluorescence complementation experiments, PAR4 formed homodimers that were disrupted by unlabeled PAR4 in a concentration-dependent manner, but not by rhodopsin. In BRET experiments, the PAR4 homodimers showed a specific interaction as indicated by a hyperbolic BRET signal in response to increasing PAR4-GFP expression. PAR4 did not interact with rhodopsin in BRET assays. The threshold maximum BRET signal was disrupted in a concentration-dependent manner by unlabeled PAR4. In contrast, rhodopsin was unable to disrupt the BRET signal, indicating that the disruption of the PAR4 homodimer is not due to nonspecific interactions. A panel of rho-PAR4 chimeras and PAR4 point mutants has mapped the dimer interface to hydrophobic residues in transmembrane helix 4. Finally, mutations that disrupted dimer formation had reduced calcium mobilization in response to the PAR4 agonist peptide. These results link the loss of dimer formation to a loss of PAR4 signaling.