The role of proline residues in the structure and function of human MT2 melatonin receptor
The role of proline residues in the structure and function of human MT2 melatonin receptor
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DOI：
10.1111/j.1600-079x.2008.00598.x
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发表时间：
2008-11-01
影响因子：
10.3
通讯作者：
Teisinger, Jan
中科院分区：
文献类型：
--
作者：
Mazna, Petr;Grycova, Lenka;Teisinger, Jan
褪黑激素是所有脊椎动物各种生理过程的重要调节剂。在哺乳动物中，两种 G 蛋白偶联褪黑激素受体 (GPCR) 介导某些褪黑激素的作用：MT1 和 MT2。大多数 GPCR 的跨膜结构域 (TM) 包含一组高度保守的脯氨酸残基，这些残基可能发挥着重要的结构和功能作用。由于 MT2 受体的 TM 片段与其他视紫红质样受体 (rGPCR) 相比，在特定脯氨酸残基的表达方面表现出一些有趣的差异，因此我们研究了脯氨酸残基在此受体的结构和功能中的作用。 MT2受体TM片段中的所有脯氨酸分别被丙氨酸和/或甘氨酸取代。此外，位于TM7中的不寻常的NAxxY基序发生突变，产生在大多数rGPCR蛋白中发现的高度保守的NPxxY基序。在 CHO-K1 细胞中瞬时表达后，分别使用 (125)I-mel 和 [(35)S]GTP gamma S 结合测定分析突变受体的结合特性及其转导信号的能力。通过嵌入完全水合磷脂双层中的 MT2 受体的分子动力学模拟来评估所进行的突变对受体结构的影响。我们的结果表明残基 P174、P212 和 P266 对于人 MT2 受体的配体结合和/或信号传导非常重要。我们还表明，TM7 中不寻常的 NAxxY 序列（突变 A305P 和 A305V）内的变化产生有缺陷的 MT2 受体，表明该基序在褪黑激素受体功能中的重要作用。
Melatonin functions as an essential regulator of various physiological processes in all vertebrate species. In mammals, two G protein-coupled melatonin receptors (GPCR) mediate some melatonin's actions: MT1 and MT2. Transmembrane domains (TM) of most GPCRs contain a set of highly conserved proline residues that presumably play important structural and functional roles. As TM segments of MT2 receptor display several interesting differences in expression of specific proline residues compared to other rhodopsin-like receptors (rGPCRs), we investigated the role of proline residues in the structure and function of this receptor. All prolines in TM segments of MT2 receptor were individually replaced with alanine and/or glycine. In addition, the unusual NAxxY motif located in TM7 was mutated to generate highly conserved NPxxY motif found in the majority of rGPCR proteins. Following transient expression in CHO-K1 cells, binding properties of the mutant receptors and their ability to transduce signals were analyzed using (125)I-mel- and [(35)S]GTP gamma S-binding assays, respectively. The impact of the performed mutations on the receptor structure was assessed by molecular dynamic simulations of MT2 receptors embedded in the fully hydrated phospholipid bilayer. Our results indicate that residues P174, P212 and P266 are important for the ligand binding and/or signaling of the human MT2 receptor. We also show that changes within the unusual NAxxY sequence in the TM7 (mutations A305P and A305V) produce defective MT2 receptors indicating an important role of this motif in the function of melatonin receptors.