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Insights into activation and dimerization of soluble guanylate cyclase

The intracellular nitric oxide receptor, soluble guanylate cyclase (sGC), is an obligate heterodimer of an α- and a smaller, heme-containing β-subunit. The prosthetic heme group is essential for the enzyme's NO-sensing capability and heme-free sGC has been shown to be unesponsive to NO. The generation of a cGMP reporter cell line together with the NO-independent but heme-dependent sGC stimulator BAY 41–2272, the NO- and heme-independent sGC activator BAY 58–2667, NO, and ODQ enabled us for the first time to distinguish between both sGC states in intact cells. Investigation of the activation profile of different sGC mutants by transient transfection into this cGMP reporter cell led to the identification of the heme binding motif Tyr135-x-Ser137-x-Arg139 in addition to the axial heme-ligand His105. Mutational analysis of the β heme-binding site identified the amino acids βAsp44, βAsp45 and βPhe74 as being crucially important for the heme-induced sGC activation.

Heterodimerization of the α/β-subunits has been proposed to be mediated mainly by the central region of sGC based on the homology of amino acids to the sequence mediating particulate guanylate cyclase homodimerization and, in addition, coprecipation studies with sGC. However, the methodical limitations of these techniques foiled the accurate identification of segments contributing to the sGC heterodimerization. To overcome these obstacles we applied the recently developed method of bimolecular fluorescence complementation (BiFC). BiFC is based on the ability of two nonfluorescent fragments of YFP to form the functional fluorophor upon interaction of the fused α1- and β1-sGC subunits. This technique enabled us to map stretches involved in the process of heterodimerization in a non-invasive manner directly in the intracellular environment. A screen of the heterodimeric profile of small deletion mutants revealed that segments spanning amino acids α1363–372, α1403–422, α1440–459 and β1212–222, β1304–333, β1344–363, β1381–400 within the predicted dimerization region are involved in the process of heterodimerization. In conclusion it is evident that the dimerization region of the sGC α and β subunit consists of discontinuous binding modules as already postulated for β1-sGC based on coprecipitation studies. Moreover, the BiFC study supports the hypothesis that the interaction of α11-sGC is at least partially mediated by an amphipathic α-helical structure. In summary, our studies demonstrate that the cGMP readout cell line in combination with the modulators of sGC activity and the recently developed method of BiFC represent powerful tools to elucidate the process of sGC dimerization and activation.

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Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution 2.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Rothkegel, C., Schmidt, P.M., Atkins, DJ. et al. Insights into activation and dimerization of soluble guanylate cyclase. BMC Pharmacol 7 (Suppl 1), S36 (2007).

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