A model for constitutive lutropin receptor activation based on molecular simulation and engineered mutations in transmembrane helices 6 and 7
A model for constitutive lutropin receptor activation based on molecular simulation and engineered mutations in transmembrane helices 6 and 7
复制标题
DOI：
10.1074/jbc.m203272200
复制
发表时间：
2002-08-30
影响因子：
4.8
通讯作者：
Puett, D
中科院分区：
文献类型：
--
作者：
Angelova, K;Fanelli, F;Puett, D
许多自然发生和工程突变导致G蛋白偶联lutropin受体（LHR）的组成性激活，其中一些也导致配体反应性降低。为了阐明这种七螺旋受体螺旋间相互作用的性质及其随激活而发生的变化，我们对大鼠LHR的跨膜螺旋6和7进行了定点诱变，制备并表征了一些单、双和三重突变体。有效的组成型激活突变体D556(6.44)H和D556(6.44)Q与较弱的激活突变体N593(7.45)R和N597(7.49)Q以及丧失响应性的突变体N593(7.45)A结合。利用一种基于视紫红质晶体结构的新受体模型，对工程突变体进行了模拟。结果表明，Asp-556（6.44）突变引起的组成性LHR激活是由野生型受体Asp-556（6.44）和Asn-593（7.45）之间相互作用的破坏或减弱引起的。虽然这种扰动是Asp-556激活突变所特有的（6.44），但所有最活跃的LHR突变体的共同特征是Arg-442（3.50）和Asp-542（6.30）之间电荷增强的h键相互作用的破坏，以及螺旋3和6的细胞质延伸的溶剂可及性增加，这可能参与受体- g蛋白界面。Asn-593（7.45）和Asn-597（7.49）似乎也是D556(6.44)H和D556(6.44)Q的高组成活性和充分的配体响应性所必需的。新的理论模型为进一步开展受体活化分子机制的实验工作奠定了基础。
Many naturally occurring and engineered mutations lead to constitutive activation of the G protein-coupled lutropin receptor (LHR), some of which also result in reduced ligand responsiveness. To elucidate the nature of interhelical interactions in this heptahelical receptor and changes thereof accompanying activation, we have utilized site-directed mutagenesis on transmembrane helices 6 and 7 of rat LHR to prepare and characterize a number of single, double, and triple mutants. The potent constitutively activating mutants, D556(6.44)H and D556(6.44)Q, were combined with weaker activating mutants, N593(7.45)R and N597(7.49)Q, and the loss-of-responsiveness mutant, N593(7.45)A. The engineered mutants have also been simulated using a new receptor model based on the crystal structure of rhodopsin. The results suggest that constitutive LHR activation by mutations at Asp-556(6.44) is triggered by the breakage or weakening of the interaction found in the wild type receptor between Asp-556(6.44) and Asn-593(7.45). Whereas this perturbation is unique to the activating mutations at Asp-556(6.44), common features to all of the most active LHR mutants are the breakage of the charge-reinforced H-bonding interaction between Arg-442(3.50) and Asp-542(6.30) and the increase in solvent accessibility of the cytosolic extensions of helices 3 and 6, which probably participate in the receptor-G protein interface. Asn-593(7.45) and Asn-597(7.49) also seem to be necessary for the high constitutive activities of D556(6.44)H and D556(6.44)Q and for full ligand responsiveness. The new theoretical model provides a foundation for further experimental work on the molecular mechanism(s) of receptor activation.