Dimerization region of soluble guanylate cyclase characterized by bimolecular fluorescence complementation in vivo
Dimerization region of soluble guanylate cyclase characterized by bimolecular fluorescence complementation in vivo
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DOI：
10.1124/mol.107.036368
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发表时间：
2007-11-01
影响因子：
3.6
通讯作者：
Stasch, Johannes-Peter
中科院分区：
文献类型：
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作者：
Rothkegel, Christiane;Schmidt, Peter M.;Stasch, Johannes-Peter
可溶性鸟苷酸环化酶（sGC）是一种广泛表达的一氧化氮（NO）受体，在信号转导中起着关键作用。NO与sGC的N-末端辅血红素部分的结合导致类似于200倍的酶活化和GTP向第二信使cGMP的转化增加。sGC作为异源二聚体存在，其二聚化主要由酶的中心区域介导。在目前的工作中，我们在预测的sGC α（1）-和β（1）-亚基的二聚化区域内构建了缺失突变体，以精确定位亚基二聚化的关键序列片段。为了跟踪突变诱导的sGC二聚化的改变，我们使用了双分子荧光互补方法，该方法允许在活细胞中以非侵入性方式可视化sGC异源二聚化。我们的研究表明跨越氨基酸α（1）363-372、α（1）403-422、α（1）440-459、β（1）212-222、β（1）304-333、β（1）344-363、和beta（1）381- 400参与异源二聚化过程，因此参与功能性sGC的表达。
The ubiquitously expressed nitric oxide (NO) receptor soluble guanylate cyclase (sGC) plays a key role in signal transduction. Binding of NO to the N-terminal prosthetic heme moiety of sGC results in similar to 200-fold activation of the enzyme and an increased conversion of GTP into the second messenger cGMP. sGC exists as a heterodimer the dimerization of which is mediated mainly by the central region of the enzyme. In the present work, we constructed deletion mutants within the predicted dimerization region of the sGC alpha(1)- and beta(1)-subunit to precisely map the sequence segments crucial for subunit dimerization. To track mutation-induced alterations of sGC dimerization, we used a bimolecular fluorescence complementation approach that allows visualizing sGC heterodimerization in a noninvasive manner in living cells. Our study suggests that segments spanning amino acids alpha(1)363-372, alpha(1)403-422, alpha(1)440-459, beta(1)212-222, beta(1)304-333, beta(1)344-363, and beta(1)381-400 within the predicted dimerization region are involved in the process of heterodimerization and therefore in the expression of functional sGC.