Identification of acidic residues in the extracellular loops of the seven-transmembrane domain of the human Ca2+ receptor critical for response to Ca2+ and a positive allosteric modulator
Identification of acidic residues in the extracellular loops of the seven-transmembrane domain of the human Ca2+ receptor critical for response to Ca2+ and a positive allosteric modulator
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DOI：
10.1074/jbc.m207100200
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发表时间：
2002-11-29
影响因子：
4.8
通讯作者：
Spiegel, AM
中科院分区：
文献类型：
--
作者：
Hu, JX;Reyes-Cruz, G;Spiegel, AM
我们研究了人类Ca2+受体（hCaR）的7跨膜结构域的胞外环（exo-loops）中的8个酸性残基在Ca2+激活受体和响应正变构调节剂NPS R-568中的作用。在全长受体和缺乏细胞外结构域的截断受体（Rho-C-hCaR）的情况下，我们将每个酸性残基突变为。研究了丙氨酸对受体表达、Ca2+激活和NPS R-568对Ca2+敏感性增强的影响。在8个酸性残基中，外显环2中的3个(Asp(758), Glu（759）和Glu（767）中的任何一个突变，都增加了全长hCaR和Rho-C-hCaR对Ca2+激活的敏感性。外环2中所有五个酸性残基的突变，无论是在全长受体还是在Rho-C-hCaR中，都会损害突变受体的细胞表面表达，从而在很大程度上消除对Ca2+的反应。外环3中Glu（837）突变为丙氨酸并没有改变全长受体的Ca2+敏感性，但在后一种情况下和Rho-C-hCaR中，Glu（837）的丙氨酸取代显著降低了对NPS R-568的敏感性。我们的数据表明，外环2中的三个特定酸性残基在Wall活化中起关键作用，外环3和跨膜螺旋7之间的Glu（837）在响应NPS R-568时起关键作用。根据这些结果，我们推测我们在外环2中发现的三个酸性残基有助于维持hCaR的七跨膜结构域的非活性构象。
We investigated the role of the eight acidic residues in the extracellular loops (exo-loops) of the seven-transmembrane domain of the human Ca2+ receptor (hCaR) in receptor activation by Ca2+ and in response to a positive allosteric modulator, NPS R-568. Both in the context of the full-length receptor and of a truncated receptor lacking the extracellular domain (Rho-C-hCaR), we mutated each acidic residue to. alanine, singly and in combination, and tested the effect on expression of the receptor, on activation by Ca2+, and on NPS R-568 augmentation of sensitivity to Ca2+. Of the eight acidic residues, mutation of any of three in exo-loop 2, Asp(758), Glu(759), and Glu(767), increased the sensitivity of both the full-length hCaR and of Rho-C-hCaR to activation by Ca2+. Mutation of all five acidic residues in exo-loop 2, whether in the full-length receptor or in Rho-C-hCaR, impaired cell surface expression of the mutant receptor and thereby largely abolished response to Ca2+. Mutation of Glu(837) in exo-loop 3 to alanine did not alter Ca2+ sensitivity of the full-length receptor, but in both the latter context and in Rho-C-hCaR, alanine substitution of Glu(837) drastically reduced sensitivity to NPS R-568. Our data point to a key role of three specific acidic residues in exo-loop 2 in Wall activation and to Glu(837) at the junction between exo-loop 3 and transmembrane helix seven in response to NPS R-568. We speculate on the basis of these results that the three acidic residues we identified in exo-loop 2 help maintain an inactive conformation of the seven-transmembrane domain of the hCaR.