Biophysical and functional characterization of the human TAS1R2 sweet taste receptor overexpressed in a HEK293S inducible cell line.
Biophysical and functional characterization of the human TAS1R2 sweet taste receptor overexpressed in a HEK293S inducible cell line.
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DOI：
10.1038/s41598-021-01731-3
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发表时间：
2021-11-15
影响因子：
4.6
通讯作者：
Briand L
中科院分区：
文献类型：
--
作者：
Belloir C;Brulé M;Tornier L;Neiers F;Briand L
甜味感知由TAS 1 R2和TAS 1 R3亚基组装形成的异二聚体受体介导。TAS 1 R2和TAS 1 R3是C类G蛋白偶联受体，其成员具有共同的拓扑结构，包括通过富含半胱氨酸的结构域连接至七跨膜结构域（TMD）的大的细胞外N末端结构域（NTD）。TAS 1 R2-NTD含有甜味化合物的主要结合位点，包括天然糖和高效甜味剂，而TAS 1 R2-TMD已显示出结合有限数量的甜味化合物。为了了解受体-配体相互作用的分子机制，我们在稳定的四环素诱导的HEK 293 S细胞系中过表达人TAS 1 R2（hTAS 1 R2），并纯化洗涤剂溶解的受体。圆二色谱研究表明，hTAS 1 R2正确折叠的二级结构的证据。使用尺寸排阻色谱结合光散射，我们发现，hTAS 1 R2亚基是一个二聚体。通过固有的色氨酸荧光来定量配体结合特性。由于技术限制，天然糖尚未进行测试。然而，我们发现hTAS 1 R2能够结合高效甜味剂，其Kd值与生理检测一致。这项研究提供了一个新的实验策略，以确定新的甜味剂或味觉调节剂，作用于hTAS 1 R2，是结构查询和生物物理研究的先决条件。
Sweet taste perception is mediated by a heterodimeric receptor formed by the assembly of the TAS1R2 and TAS1R3 subunits. TAS1R2 and TAS1R3 are class C G-protein-coupled receptors whose members share a common topology, including a large extracellular N-terminal domain (NTD) linked to a seven transmembrane domain (TMD) by a cysteine-rich domain. TAS1R2-NTD contains the primary binding site for sweet compounds, including natural sugars and high-potency sweeteners, whereas the TAS1R2-TMD has been shown to bind a limited number of sweet tasting compounds. To understand the molecular mechanisms governing receptor–ligand interactions, we overexpressed the human TAS1R2 (hTAS1R2) in a stable tetracycline-inducible HEK293S cell line and purified the detergent-solubilized receptor. Circular dichroism spectroscopic studies revealed that hTAS1R2 was properly folded with evidence of secondary structures. Using size exclusion chromatography coupled to light scattering, we found that the hTAS1R2 subunit is a dimer. Ligand binding properties were quantified by intrinsic tryptophan fluorescence. Due to technical limitations, natural sugars have not been tested. However, we showed that hTAS1R2 is capable of binding high potency sweeteners with Kd values that are in agreement with physiological detection. This study offers a new experimental strategy to identify new sweeteners or taste modulators that act on the hTAS1R2 and is a prerequisite for structural query and biophysical studies.
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影响因子：
64.8
作者：
通讯作者：
--
影响因子：
13.8
作者：
Ellaithy A;Gonzalez-Maeso J;Logothetis DA;Levitz J
通讯作者：
Levitz J
影响因子：
4.6
作者：
Assadi-Porter, Fariba M.;Radek, James;Tonelli, Marco
通讯作者：
Tonelli, Marco
DOI：
10.1016/j.bbamem.2009.07.021
发表时间：
2010-02
期刊：
Biochimica et biophysica acta
影响因子：
--
作者：
Assadi-Porter FM;Tonelli M;Maillet EL;Markley JL;Max M
通讯作者：
Max M
影响因子：
64.8
作者：
Kang Y;Zhou XE;Gao X;He Y;Liu W;Ishchenko A;Barty A;White TA;Yefanov O;Han GW;Xu Q;de Waal PW;Ke J;Tan MH;Zhang C;Moeller A;West GM;Pascal BD;Van Eps N;Caro LN;Vishnivetskiy SA;Lee RJ;Suino-Powell KM;Gu X;Pal K;Ma J;Zhi X;Boutet S;Williams GJ;Messerschmidt M;Gati C;Zatsepin NA;Wang D;James D;Basu S;Roy-Chowdhury S;Conrad CE;Coe J;Liu H;Lisova S;Kupitz C;Grotjohann I;Fromme R;Jiang Y;Tan M;Yang H;Li J;Wang M;Zheng Z;Li D;Howe N;Zhao Y;Standfuss J;Diederichs K;Dong Y;Potter CS;Carragher B;Caffrey M;Jiang H;Chapman HN;Spence JC;Fromme P;Weierstall U;Ernst OP;Katritch V;Gurevich VV;Griffin PR;Hubbell WL;Stevens RC;Cherezov V;Melcher K;Xu HE
通讯作者：
Xu HE