Two distinct calmodulin binding sites in the third intracellular loop and carboxyl tail of angiotensin II (AT(1A)) receptor.
Two distinct calmodulin binding sites in the third intracellular loop and carboxyl tail of angiotensin II (AT(1A)) receptor.
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DOI：
10.1371/journal.pone.0065266
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Yang Q
中科院分区：
文献类型：
--
作者：
Zhang R;Liu Z;Qu Y;Xu Y;Yang Q
在这项研究中，我们提出的数据支持存在两个不同的钙调素结合位点的血管紧张素II受体（AT 1A），在质膜区域的N-末端的第三个细胞内环（i3，氨基酸214-231）和羧基尾的受体（ct，302-317）。我们使用生物发光共振能量转移测定记录钙调素与AT 1A全受体和GST融合蛋白下拉的相互作用，以证明i3和ct以Ca 2+依赖的方式与钙调素相互作用。前者属于1-12模体，后者属于1-5-10钙调素结合模体。通过丹磺酰-钙调素荧光评估，钙调素对i3的表观Kd为177.0±9.1 nM，对ct的表观Kd为79.4±7.9 nM。替换色氨酸（W219）为丙氨酸在i3中，和苯丙氨酸（F309或F313）为丙氨酸在ct中降低了它们的钙调蛋白的结合亲和力，如通过计算机对接模拟预测的。外源性钙调素减弱了G蛋白βγ亚基与i3和ct之间的相互作用，对ct的作用比i3更明显。突变W219 A、F309 A和F313 A没有改变Gβγ结合，但降低了钙调蛋白与Gβγ竞争的能力，表明钙调蛋白和Gβγ对i3和ct的结合要求重叠，但不相同。钙调素干扰Gβγ与AT 1A受体i3和ct区域的结合，强烈表明钙调素在调节受体的Gβγ依赖性信号传导中起关键作用。
In this study, we present data that support the presence of two distinct calmodulin binding sites within the angiotensin II receptor (AT1A), at juxtamembrane regions of the N-terminus of the third intracellular loop (i3, amino acids 214–231) and carboxyl tail of the receptor (ct, 302–317). We used bioluminescence resonance energy transfer assays to document interactions of calmodulin with the AT1A holo-receptor and GST-fusion protein pull-downs to demonstrate that i3 and ct interact with calmodulin in a Ca2+-dependent fashion. The former is a 1–12 motif and the latter belongs to 1-5-10 calmodulin binding motif. The apparent Kd of calmodulin for i3 is 177.0±9.1 nM, and for ct is 79.4±7.9 nM as assessed by dansyl-calmodulin fluorescence. Replacement of the tryptophan (W219) for alanine in i3, and phenylalanine (F309 or F313) for alanine in ct reduced their binding affinities for calmodulin, as predicted by computer docking simulations. Exogenously applied calmodulin attenuated interactions between G protein βγ subunits and i3 and ct, somewhat more so for ct than i3. Mutations W219A, F309A, and F313A did not alter Gβγ binding, but reduced the ability of calmodulin to compete with Gβγ, suggesting that calmodulin and Gβγ have overlapping, but not identical, binding requirements for i3 and ct. Calmodulin interference with the Gβγ binding to i3 and ct regions of the AT1A receptor strongly suggests that calmodulin plays critical roles in regulating Gβγ-dependent signaling of the receptor.
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影响因子：
2.1
作者：
Ishikawa, K;Nash, SR;Nakanishi, S
通讯作者：
Nakanishi, S
影响因子：
5.6
作者：
BABU, YS;BUGG, CE;COOK, WJ
通讯作者：
COOK, WJ
影响因子：
64.8
作者：
Ango, F;Prézeau, L;Fagni, L
通讯作者：
Fagni, L
DOI：
10.1523/jneurosci.6253-10.2011
发表时间：
2011-04-20
期刊：
The Journal of neuroscience : the official journal of the Society for Neuroscience
影响因子：
--
作者：
Choi KY;Chung S;Roche KW
通讯作者：
Roche KW
影响因子：
3.5
作者：
Airas, JM;Betz, H;El Far, O
通讯作者：
El Far, O