Identification of Critical Residues in the Carboxy Terminus of the Dopamine Transporter Involved in the G Protein βγ-Induced Dopamine Efflux.
Identification of Critical Residues in the Carboxy Terminus of the Dopamine Transporter Involved in the G Protein βγ-Induced Dopamine Efflux.
复制标题
DOI：
10.3389/fphar.2021.642881
复制
发表时间：
2021
影响因子：
5.6
通讯作者：
Torres GE
中科院分区：
文献类型：
--
作者：
Pino JA;Nuñez-Vivanco G;Hidalgo G;Reyes Parada M;Khoshbouei H;Torres GE
多巴胺转运蛋白（DAT）通过将多巴胺（DA）重摄取回突触前末梢，在调节脑内DA稳态中起着至关重要的作用。除了再摄取，DAT还能够通过称为DAT介导的DA流出的过程释放DA。这是一种机制，通过这种机制，强效和高度成瘾的精神兴奋剂，如安非他明（AMPH）及其类似物，增加了大脑动机和奖励区域的细胞外DA水平。最近，我们发现G蛋白βγ亚基（Gβγ）与DAT结合，并且Gβγ的活化导致DAT介导的外排-与AMPH类似的机制。之前，我们已经证明Gβγ直接结合DAT细胞内羧基末端的15个残基（残基582-596）。此外，含有DAT残基582 - 596的达特肽能够阻断Gβγ通过DAT诱导的DA外排。在这里，我们使用计算生物学、诱变、生物化学和功能测定的组合来鉴定DAT羧基末端582-596序列内参与DAT-G βγ相互作用和Gβγ诱导的DA外排的氨基酸残基。我们的计算机蛋白质-蛋白质对接分析预测了F587和R588残基在与Gβγ残基相互作用网络中的重要性。此外，我们观察到将R588突变为丙氨酸残基导致突变的DAT，其表现出由Gβγ激活引起的DA流出减弱。我们证明位于DAT羧基端的R588和较小程度上的F5837在DAT-G βγ物理相互作用和促进DA流出中起关键作用。这些结果确定了一个潜在的新的药理学靶点，用于治疗涉及DAT功能的神经精神疾病，包括ADHD和物质使用障碍。
The dopamine transporter (DAT) plays a crucial role in the regulation of brain dopamine (DA) homeostasis through the re-uptake of DA back into the presynaptic terminal. In addition to re-uptake, DAT is also able to release DA through a process referred to as DAT-mediated DA efflux. This is the mechanism by which potent and highly addictive psychostimulants, such as amphetamine (AMPH) and its analogues, increase extracellular DA levels in motivational and reward areas of the brain. Recently, we discovered that G protein βγ subunits (Gβγ) binds to the DAT, and that activation of Gβγ results in DAT-mediated efflux - a similar mechanism as AMPH. Previously, we have shown that Gβγ binds directly to a stretch of 15 residues within the intracellular carboxy terminus of DAT (residues 582–596). Additionally, a TAT peptide containing residues 582 to 596 of DAT was able to block the Gβγ-induced DA efflux through DAT. Here, we use a combination of computational biology, mutagenesis, biochemical, and functional assays to identify the amino acid residues within the 582–596 sequence of the DAT carboxy terminus involved in the DAT-Gβγ interaction and Gβγ-induced DA efflux. Our in-silico protein-protein docking analysis predicted the importance of F587 and R588 residues in a network of interactions with residues in Gβγ. In addition, we observed that mutating R588 to alanine residue resulted in a mutant DAT which exhibited attenuated DA efflux induced by Gβγ activation. We demonstrate that R588, and to a lesser extent F5837, located within the carboxy terminus of DAT play a critical role in the DAT-Gβγ physical interaction and promotion of DA efflux. These results identify a potential new pharmacological target for the treatment of neuropsychiatric conditions in which DAT functionality is implicated including ADHD and substance use disorder.
登录
查看更多内容
影响因子：
64.8
作者：
Koehl A;Hu H;Maeda S;Zhang Y;Qu Q;Paggi JM;Latorraca NR;Hilger D;Dawson R;Matile H;Schertler GFX;Granier S;Weis WI;Dror RO;Manglik A;Skiniotis G;Kobilka BK
通讯作者：
Kobilka BK
影响因子：
5.3
作者：
Gabriel, Luke R.;Wu, Sijia;Melikian, Haley E.
通讯作者：
Melikian, Haley E.
影响因子：
25
作者：
Holton, KL;Loder, MK;Melikian, HE
通讯作者：
Melikian, HE
影响因子：
14.8
作者：
Kozakov D;Hall DR;Xia B;Porter KA;Padhorny D;Yueh C;Beglov D;Vajda S
通讯作者：
Vajda S
影响因子：
4.8
作者：
Johnson, LA;Guptaroy, B;Gnegy, ME
通讯作者：
Gnegy, ME