Functional fusions of T4 lysozyme in the third intracellular loop of a G protein-coupled receptor identified by a random screening approach in yeast.
Functional fusions of T4 lysozyme in the third intracellular loop of a G protein-coupled receptor identified by a random screening approach in yeast.
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通过酵母中的随机筛选方法鉴定出 T4 溶菌酶在 G 蛋白偶联受体第三个细胞内环中的功能融合。
DOI：
10.1093/protein/gzs070
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
Dumont,MarkE
中科院分区：
文献类型：
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作者：
Mathew,Elizabeth;Ding,Fa-Xiang;Naider,Fred;Dumont,MarkE
将稳定的可溶性蛋白插入跨膜蛋白的环中已被证明是增强其稳定性和结晶的成功方法，并且在插入的蛋白可以调节或报告膜蛋白活性的情况下也可能是有用的。虽然使用T4溶菌酶来替换G蛋白偶联受体（GPCR）的第三胞内环的部分已经允许确定这类重要受体的成员的结构，但是这种融合蛋白的产生通常导致所得融合蛋白的信号传导功能的丧失，因为GPCR的第三胞内环在它们与G蛋白的相互作用中起关键作用。我们在这里描述了一种随机筛选方法，允许T4溶菌酶插入到酵母α-信息素受体的第三环的不同位置，由酵母STE 2基因编码的GPCR。插入伴随着不同程度的缺失或重复的环。一组表型筛选允许检测潜在的罕见变体受体，所述受体被表达、结合激动剂并且能够通过同源G蛋白的活化进行信号转导。在插入的T4溶菌酶的任一侧上含有至少部分重复的环的大部分筛选的全长受体变体保留响应于配体结合而激活下游信号传导途径的能力。然而，我们无法确定任何受体与截短的C-末端，保留显着的信号转导功能的存在下，插入T4溶菌酶。我们的研究结果建立了在其第三胞内环中插入T4溶菌酶的功能受体的可行性。
The insertion of a stable soluble protein into loops of transmembrane proteins has proved to be a successful approach for enhancing their stabilities and crystallization, and may also be useful in contexts where the inserted proteins can modulate or report on the activities of membrane proteins. While the use of T4 lysozyme to replace portions of the third intracellular loops of G protein-coupled receptors (GPCRs) has allowed determination of the structures of members of this important class of receptors, the creation of such fusion proteins generally leads to loss of signaling function of the resulting fusion protein, since the third intracellular loops of GPCRs play critical roles in their interactions with G proteins. We describe here a random screening approach allowing insertion of T4 lysozyme into diverse positions in the third loop of the yeast α-pheromone receptor, a GPCR encoded by the yeastSTE2gene. Insertions were accompanied by varying extents of deletion or duplication of the loop. A set of phenotypic screens allow detection of potentially rare variant receptors that are expressed, bind to agonist and are capable of signal transduction via activation of the cognate G protein. A large fraction of screened full-length receptor variants containing at least partial duplications of the loop on either side of the inserted T4 lysozyme retain the ability to activate the downstream signaling pathway in response to binding of ligand. However, we were unable to identify any receptors with truncated C-termini that retain significant signaling function in the presence of inserted T4 lysozyme. Our results establish the feasibility of creating functional receptors containing insertions of T4 lysozyme in their third intracellular loops.