Conformational Toggle Switches Implicated in Basal Constitutive and Agonist-Induced Activated States of 5-Hydroxytryptamine-4 Receptors
Conformational Toggle Switches Implicated in Basal Constitutive and Agonist-Induced Activated States of 5-Hydroxytryptamine-4 Receptors
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DOI：
10.1124/mol.108.053686
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发表时间：
2009-04-01
影响因子：
3.6
通讯作者：
Pardo, Leonardo
中科院分区：
文献类型：
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作者：
Pellissier, Lucie P.;Sallander, Jessica;Pardo, Leonardo
扩展的经典三元复合物模型预测，G蛋白偶联受体（GPCR）只存在两种相互转换的状态：一个无活性的R和一个活性的R*。然而，除了沉默的非活性R基态（Rg）之外，还可以存在不同结构的活性R* 复合物。在这里，我们证明，在细胞的情况下，几个R* 状态的5-羟色胺-4（5-HT 4）受体涉及不同的侧链构象切换开关。使用定点突变和分子模拟方法，我们表明，基础组成型受体（R* 基础）的结果从一个强制性的双拨动开关（Thr3.36从非活性g-到活性g+和Trp6.48从非活性g-到活性t）的稳定。苏氨酸或色氨酸突变为丙氨酸导致与Rg类似的R* 基础活性降低。T3.36A突变表明，Thr3.36开关在5-HT诱导的R*（R*-5-HT）和BIMU 8诱导的R* 稳定中起次要作用。（R*-BIMU 8），并且在由（S）-扎考必利，西沙必利，和1-（4-氨基-5-氯-2-甲氧基苯基）-3-（1-丁基-4-哌啶基）-1-丙酮（RS 67333）（R*-苯甲酰胺）。因此，苯甲酰胺通过与活性g+构象中的Thr3.36形成特异性氢键来稳定R*-苯甲酰胺。相反，R*-BIMU 8可能是Trp6.48通过该残基与BIMU 8的羧基的氢键作用从无活性g+直接构象转变为活性t的结果。令我们惊讶的是，Trp6.48拨动开关不是天然激动剂5-HT激活受体所必需的。R*-5-HT可能通过其他活化途径获得。因此，在R* basal、R*-5-HT、R*-苯甲酰胺和R*-BIMU 8的稳定化期间发生不同的构象排列。
The extended classic ternary complex model predicts that a G protein-coupled receptor (GPCR) exists in only two interconvertible states: an inactive R, and an active R*. However, different structural active R* complexes may exist in addition to a silent inactive R ground state (Rg). Here we demonstrate, in a cellular context, that several R* states of 5-hydroxytryptamine-4 (5-HT4) receptors involve different side-chain conformational toggle switches. Using site-directed mutagenesis and molecular modeling approaches, we show that the basal constitutive receptor (R* basal) results from stabilization of an obligatory double toggle switch (Thr3.36 from inactive g-to active g+ and Trp6.48 from inactive g-to active t). Mutation of either threonine or tryptophan to alanine resulted in a lowering of the activity of the R* basal similar to the Rg. The T3.36A mutation shows that the Thr3.36 toggle switch plays a minor role in the stabilization of R* induced by 5-HT (R*-5-HT) and BIMU8 (R*-BIMU8) and is fully required in the stabilization of R* induced by (S)-zacopride, cisapride, and 1-(4-amino-5-chloro-2-methoxyphenyl)-3-(1-butyl-4-piperidinyl)-1-propanone (RS 67333) (R*-benzamides). Thus, benzamides stabilize R*-benzamides by forming a specific hydrogen bond with Thr3.36 in the active g+ conformation. Conversely, R*-BIMU8 was probably the result of a direct conformational transition of Trp6.48 from inactive g+ to active t by hydrogen bonding of this residue to a carboxyl group of BIMU8. We were surprised that the Trp6.48 toggle switch was not necessary for receptor activation by the natural agonist 5-HT. R*-5-HT is probably attained through other routes of activation. Thus, different conformational arrangements occur during stabilization of R* basal, R*-5-HT, R*-benzamides, and R*-BIMU8.