Gain-of-function screen of α-transducin identifies an essential phenylalanine residue necessary for full effector activation
Gain-of-function screen of α-transducin identifies an essential phenylalanine residue necessary for full effector activation
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DOI：
10.1074/jbc.ra118.003746
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发表时间：
2018-11-16
影响因子：
4.8
通讯作者：
Ramachandran, Sekar
中科院分区：
文献类型：
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作者：
Milano, Shawn K.;Wang, Chenyue;Ramachandran, Sekar
异源三聚体GTP结合蛋白（G蛋白）的亚基上的两个区域，开关II/2螺旋（其在GDP-GTP交换时改变构象）和3螺旋，已被证明含有其效应蛋白的结合位点。然而，G亚基与其效应蛋白的结合如何转化为效应活性的刺激仍然知之甚少。在这里，我们利用重建的视紫红质耦合光转导系统来解决这个问题，并确定了一个独特的表面和一个必要的残基上的G蛋白转导（（T））的亚基，这是必要的，以充分激活其效应酶，cGMP磷酸二酯酶（PDE）。我们从嵌合G蛋白亚基（（T）*）开始，其包含主要来自（T）的残基和来自G（i1）亚基（（i1））的一小段残基，其仅微弱地刺激PDE活性。然后，我们通过系统地替换（T）* 内相应的（i1）残基来恢复（T）残基，目的是完全恢复PDE刺激活性。这些实验表明，G/4环和位置283处的苯丙氨酸残基对于赋予（T）* 亚基完整的PDE刺激能力是必需的。我们进一步证明了G（s）蛋白亚基内的相同区域和氨基酸对于腺苷酸环化酶的完全激活是必需的。这些发现强调了G/4环和G亚基（T）和（S）上该区域内的必需苯丙氨酸残基的重要性，因为它们对于效应子活性的选择性和最佳刺激是关键的。
Two regions on the subunits of heterotrimeric GTP-binding proteins (G-proteins), the Switch II/2 helix (which changes conformation upon GDP-GTP exchange) and the 3 helix, have been shown to contain the binding sites for their effector proteins. However, how the binding of G subunits to their effector proteins is translated into the stimulation of effector activity is still poorly understood. Here, we took advantage of a reconstituted rhodopsin-coupled phototransduction system to address this question and identified a distinct surface and an essential residue on the subunit of the G-protein transducin ((T)) that is necessary to fully activate its effector enzyme, the cGMP phosphodiesterase (PDE). We started with a chimeric G-protein subunit ((T)*) comprising residues mainly from (T) and a short stretch of residues from the G(i1) subunit ((i1)), which only weakly stimulates PDE activity. We then reinstated the (T) residues by systematically replacing the corresponding (i1) residues within (T)* with the aim of fully restoring PDE stimulatory activity. These experiments revealed that the G/4 loop and a phenylalanine residue at position 283 are essential for conferring the (T)* subunit with full PDE stimulatory capability. We further demonstrated that this same region and amino acid within the subunit of the G(s) protein ((s)) are necessary for full adenylyl cyclase activation. These findings highlight the importance of the G/4 loop and of an essential phenylalanine residue within this region on G subunits (T) and (s) as being pivotal for their selective and optimal stimulation of effector activity.