Modulation of interprotomer relationships is important for activation of dimeric calcium-sensing receptor
Modulation of interprotomer relationships is important for activation of dimeric calcium-sensing receptor
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DOI：
10.1074/jbc.m307422200
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发表时间：
2004-04-02
影响因子：
4.8
通讯作者：
Bai, M
中科院分区：
文献类型：
--
作者：
Jiang, YF;Minet, E;Bai, M
细胞外钙敏感受体（CaR）通过其N-末端胞外结构域（ECD）内的半胱氨酸残基形成二硫键连接的二聚体。然而，这些二硫键对于二聚体CaR的形成和两个失活汽车的功能重建是不利的。在这项研究中，使用分子建模，诱变，生物化学和生物物理分析，我们研究了两个亮氨酸残基，Leu-112和Leu-156，在ECD的CaR的非共价二聚化和功能重建的重要性。我们发现携带L112 S和L156 S的突变受体仍然主要以共价连接的二聚体形式存在，并且对钙的表观亲和力比野生型受体显著更高。然而，L112 S、L156 S、C129 S和C131 S四种突变的组合显著降低受体二聚化并显著灭活CaR。我们还发现，L112 S和L156 S介导的非共价分子间相互作用的重要功能重建。因为突变两个半胱氨酸或两个亮氨酸增强了CaR的表观配体亲和力，所以很可能由这些亮氨酸和半胱氨酸连接的两个受体原体之间的分子间关系的变化对于受体活化是必不可少的。此外，这些突变不太可能对二聚体受体的每个原聚体的二级结构具有负面影响。因此，组合突变对CaR功能的不利影响进一步表明，通过其ECD的CaR二聚化对于形成CaR的功能性三级结构是必不可少的。
The extracellular calcium-sensing receptor (CaR) forms a disulfide-linked dimer through cysteine residues within its N-terminal extracellular domain (ECD). However, these disulfide linkages are dispensable for the formation of the dimeric CaR and for the functional reconstitution of two inactive CaRs. In this study, using molecular modeling, mutagenesis, and biochemical and biophysical analyses, we examined the importance of two leucine residues, Leu-112 and Leu-156, in the ECD of the CaR for the non-covalent dimerization and functional reconstitution. We found that the mutant receptor carrying L112S and L156S still exists mostly as a covalently linked dimer and has a significantly higher apparent affinity for calcium than the wild-type receptor. However, a combination of four mutations, L112S, L156S, C129S, and C131S, significantly reduces receptor dimerization and markedly inactivates the CaR. We also found that L112S and L156S mediate the non-covalent intermolecular interactions important for functional reconstitution. Because mutating either the two cysteines or the two leucines enhances the apparent ligand affinity of the CaR, it is likely that the changes in intermolecular relationships between two receptor protomers linked by these leucines and cysteines are essential for receptor activation. Moreover, these mutations are unlikely to have negative effects on the secondary structure of each protomer of the dimeric receptor. Thus, the detrimental effects of the combined mutations on the function of the CaR further suggest that CaR dimerization through its ECD is essential for the formation of a functional tertiary structure of the CaR.