Dimerization of cAMP phosphodiesterase-4 (PDE4) in living cells requires interfaces located in both the UCR1 and catalytic unit domains.
Dimerization of cAMP phosphodiesterase-4 (PDE4) in living cells requires interfaces located in both the UCR1 and catalytic unit domains.
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DOI：
10.1016/j.cellsig.2014.12.009
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发表时间：
2015-04
影响因子：
4.8
通讯作者：
Houslay MD
中科院分区：
文献类型：
--
作者：
Bolger GB;Dunlop AJ;Meng D;Day JP;Klussmann E;Baillie GS;Adams DR;Houslay MD
PDE 4家族cAMP磷酸二酯酶通过靶向cAMP分解在确定区室化cAMP信号传导中起关键作用。表达广泛发现的PDE 4D 5亚型，作为酵母双杂交系统中的诱饵和猎物，我们证明了与长PDE 4亚型形成二聚体的概念一致的相互作用。四个潜在的二聚化位点被发现使用扫描肽阵列方法，其中重组纯化的PDE 4D 5融合蛋白被用于探测覆盖整个PDE 4D 5序列的重叠肽的25-mer文库。使用由丙氨酸扫描肽阵列方法指导的定点诱变程序来定义参与PDE 4D 5二聚化的关键残基。稳定PDE 4D 5二聚化的关键残基定义在长而非短PDE 4同种型中发现的调节UCR 1区域内，即Arg 173、Asn 174和Asn 175（DD 1）簇。DD 1簇的破坏本身不足以使PDE 4D 5同源二聚体不稳定。相反，还需要破坏位于PDE 4催化单元上的额外界面以将PDE 4D 5转化为单体形式。保守的PDE 4催化单元上的第二个二聚化位点取决于关键的离子对相互作用。这涉及PDE 4D 5中的Asp 463和Arg 499，它们以反式方式相互作用，涉及参与同二聚体的两个PDE 4D 5分子。PDE 4长同种型在活细胞中采用二聚体状态，其由两个关键的贡献相互作用支撑，一个涉及UCR模块，另一个涉及核心催化结构域上的界面。我们建议，短的形式不采用二聚体的配置，因为在没有UCR 1模块，剩余的核心催化结构域接口的剩余接合提供了不足的自由能来驱动二聚化。由于四级结构的这种差异，PDE 4长型和短型的功能因此注定是固有的不同。在酵母双杂交系统中，我们发现长PDE 4亚型二聚化。扫描肽阵列和诱变定位两个二聚化表面。一个表面映射到仅在长形式中发现的调节UCR 1区域。第二个位于核心催化结构域。PDE 4长型和短型在四级结构上不同。
PDE4 family cAMP phosphodiesterases play a pivotal role in determining compartmentalised cAMP signalling through targeted cAMP breakdown. Expressing the widely found PDE4D5 isoform, as both bait and prey in a yeast 2-hybrid system, we demonstrated interaction consistent with the notion that long PDE4 isoforms form dimers. Four potential dimerization sites were uncovered using a scanning peptide array approach, where a recombinant purified PDE4D5 fusion protein was used to probe a 25-mer library of overlapping peptides covering the entire PDE4D5 sequence. Key residues involved in PDE4D5 dimerization were defined using a site-directed mutagenesis programme directed by an alanine scanning peptide array approach. Critical residues stabilising PDE4D5 dimerization were defined within the regulatory UCR1 region found in long, but not short, PDE4 isoforms, namely the Arg173, Asn174 and Asn175 (DD1) cluster. Disruption of the DD1 cluster was not sufficient, in itself, to destabilise PDE4D5 homodimers. Instead, disruption of an additional interface, located on the PDE4 catalytic unit, was also required to convert PDE4D5 into a monomeric form. This second dimerization site on the conserved PDE4 catalytic unit is dependent upon a critical ion pair interaction. This involves Asp463 and Arg499 in PDE4D5, which interact in a trans fashion involving the two PDE4D5 molecules participating in the homodimer. PDE4 long isoforms adopt a dimeric state in living cells that is underpinned by two key contributory interactions, one involving the UCR modules and one involving an interface on the core catalytic domain. We propose that short forms do not adopt a dimeric configuration because, in the absence of the UCR1 module, residual engagement of the remaining core catalytic domain interface provides insufficient free energy to drive dimerization. The functioning of PDE4 long and short forms is thus poised to be inherently distinct due to this difference in quaternary structure. In a yeast 2-hybrid system we show that long PDE4 isoforms dimerize. Scanning peptide array and mutagenesis located two dimerization surfaces. One surface maps to the regulatory UCR1 region found only in long forms. A second locates to the core catalytic domain. PDE4 long and short forms differ in quaternary structure.
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影响因子：
5.7
作者：
Card, GL;England, BP;Zhang, KYJ
通讯作者：
Zhang, KYJ
影响因子：
4.1
作者：
Bolger, Graeme B.;Baillie, George S.;Houslay, Miles D.
通讯作者：
Houslay, Miles D.
影响因子：
4.1
作者：
Baillie, George S.;Adams, David R.;Houslay, Miles D.
通讯作者：
Houslay, Miles D.
影响因子：
7.3
作者：
Baillie, GS;MacKenzie, SJ;Houslay, MD
通讯作者：
Houslay, MD
影响因子：
4.8
作者：
Hundsrucker, Christian;Skroblin, Philipp;Klussmann, Enno
通讯作者：
Klussmann, Enno