The Venus fly trap domain of the extracellular Ca2+-sensing receptor is required for L-amino acid sensing
The Venus fly trap domain of the extracellular Ca2+-sensing receptor is required for L-amino acid sensing
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DOI：
10.1074/jbc.m406164/200
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发表时间：
2004-12-10
影响因子：
4.8
通讯作者：
Conigrave, AD
中科院分区：
文献类型：
--
作者：
Mun, HC;Franks, AH;Conigrave, AD
我们先前证明了人钙敏感受体（CaR）被L-氨基酸变构激活（Conigrave，A. D、Quinn，S. J.，和Brown，E. M.等人（2000）Proc. Acad. Sci.联合S. A. 97，4814-4819）。然而，基于结构域的氨基酸结合的位置一直是不确定的。我们现在表明，金星飞陷阱（VFT）结构域的钙，但没有其他主要领域，是必需的氨基酸传感。当在HEK 293细胞中表达时，几种构建体是信息性的。首先，野生型CaR表现出变构激活的L-氨基酸如前所述。第二，两个CaR-mGlu嵌合受体结构，保留了VFT结构域的CaR，一个含有细胞外的Cys丰富的区域的CaR和其他含有Cys丰富的区域的大鼠代谢型谷氨酸1型（mGlu-1）受体，连同大鼠mGlu-1跨膜区和C-末端尾，保留了氨基酸传感。第三，CaR缺乏N-末端细胞外头部的残基1-599，但保留完整的CaR跨膜区和功能性但截短的C末端（无头-T903 CaR）未能响应L-氨基酸，但保留对II型钙模拟物CaR-467的响应性。最后，保留完整N末端的T903 CaR对照也保留L-氨基酸传感。两者合计，数据表明CaR的VFT结构域对于L-氨基酸传感是必需的，并且与VFT结构域是L-氨基酸结合位点的假设一致。这一发现支持了这样的概念，即mGlu-1的氨基酸结合位点的L-谷氨酸是保守的L-氨基酸结合位点在其同系物，钙受体。
We previously demonstrated that the human calcium-sensing receptor (CaR) is allosterically activated by L-amino acids (Conigrave, A. D., Quinn, S. J., and Brown, E. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 4814-4819). However, the domain-based location of amino acid binding has been uncertain. We now show that the Venus Fly Trap (VFT) domain of CaR, but none of its other major domains, is required for amino acid sensing. Several constructs were informative when expressed in HEK293 cells. First, the wild-type CaR exhibited allosteric activation by L-amino acids as previously observed. Second, two CaR-mGlu chimeric receptor constructs that retained the VFT domain of CaR, one containing the extracellular Cys-rich region of CaR and the other containing the Cys-rich region of the rat metabotropic glutamate type-1 (mGlu-1) receptor, together with the rat mGlu-1 transmembrane region and C-terminal tail, retained amino acid sensing. Third, a CaR lacking residues 1-599 of the N-terminal extracellular head but retaining an intact CaR transmembrane region and a functional but truncated C terminus (headless-T903 CaR) failed to respond to L-amino acids but retained responsiveness to the type-II calcimimetic NPS R-467. Finally, a T903 CaR control that retained an intact N terminus also retained L-amino acid sensing. Taken together, the data indicate that the VFT domain of CaR is necessary for L-amino acid sensing and are consistent with the hypothesis that the VFT domain is the site of L-amino acid binding. The findings support the concept that the mGlu-1 amino acid binding site for L-glutamate is conserved as an L-amino acid binding site in its homolog, the CaR.