POSTER PRESENTATION Open Access G-protein regulator LGN inhibits the activity of soluble guanylyl cyclase Swati Chauhan, Iraida Sharina, Emil Martin * From 5th International Conference on cGMP: Generators, Effectors and Therapeutic Implications Halle, Germany. 24-26 June 2011 Background Nitric oxide binds with the heme moiety of sGC and activates sGC several hundred folds the cGMP-forming activity of the enzyme. Although nitric oxide is the major physiological regulator of sGC function, the extent of this activation is influenced by a number of cellular factors, e.g. ATP, substrate GTP, reaction pro- ducts, intracellular calcium, etc. Several proteins, such as HSP70, HSP90 and CCTh, where shown to interact with sGC and modulate its function. Results In this report we provide evidence that demonstrate inhibition of sGC by a LGN-dependent mechanism. LGN, a Leu-Gly-Asn repeat enriched protein, is a known guanosine diphosphate (GDP) dissociation inhi- bitor, which regulates the Gai family of G proteins. Using the yeast two-hybrid screening, we identified LGN as a protein that interacts with both a1 and b1 subunits of sGC. This interaction was confirmed by co- immunoprecipitation of sGC and LGN proteins from the lysate of BE2 human neuroblastoma cell line. Tran- sient overexpression of LGN in sGC expressing MDA468 breast cancer cell line markedly decreased the activity of sGC in cell lysates in a gene-dose dependent fashion. On the contrary, inhibition of LGN expression by targeted siRNA increased sGC activity in the cellular lysates. In vitro pull-down experiments performed with truncated versions of LGN and sGC mapped the domains required for interaction between LGN and sGC. However, when the effect of purified LGN protein on the activity of purified sGC was tested in vitro, we found no evidence of inhibition, suggesting that LGN does not directly affect sGC function. When the same experiment was performed in the lysate of COS7 cell, we observed a marked inhibition of sGC. Conclusion These data suggest that LGN may function as an adap- tor protein for additional cellular factors inhibiting sGC. Since LGN is a known regulator of G-protein, which associates with Ga subunit, it may function as an adap- tor protein for a complex consisting of sGC, LGN and Ga, thus connecting the NO-dependent and G-protein signaling pathways. Published: 1 August 2011 doi:10.1186/1471-2210-11-S1-P15 Cite this article as: Chauhan et al.: G-protein regulator LGN inhibits the activity of soluble guanylyl cyclase. BMC Pharmacology 2011 11(Suppl 1): P15. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit * Correspondence: [email protected] University of Texas Houston Medical School, Department of Internal Medicine, Division of Cardiology, Houston, Texas, USA Chauhan et al. BMC Pharmacology 2011, 11(Suppl 1):P15 http://www.biomedcentral.com/1471-2210/11/S1/P15 © 2011 Chauhan et al; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

G-protein regulator LGN inhibits the activity of soluble guanylyl cyclase

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POSTER PRESENTATION Open Access

G-protein regulator LGN inhibits the activity ofsoluble guanylyl cyclaseSwati Chauhan, Iraida Sharina, Emil Martin*

From 5th International Conference on cGMP: Generators, Effectors and Therapeutic ImplicationsHalle, Germany. 24-26 June 2011

BackgroundNitric oxide binds with the heme moiety of sGC andactivates sGC several hundred folds the cGMP-formingactivity of the enzyme. Although nitric oxide is themajor physiological regulator of sGC function, theextent of this activation is influenced by a number ofcellular factors, e.g. ATP, substrate GTP, reaction pro-ducts, intracellular calcium, etc. Several proteins, suchas HSP70, HSP90 and CCTh, where shown to interactwith sGC and modulate its function.

ResultsIn this report we provide evidence that demonstrateinhibition of sGC by a LGN-dependent mechanism.LGN, a Leu-Gly-Asn repeat enriched protein, is aknown guanosine diphosphate (GDP) dissociation inhi-bitor, which regulates the Gai family of G proteins.Using the yeast two-hybrid screening, we identifiedLGN as a protein that interacts with both a1 and b1subunits of sGC. This interaction was confirmed by co-immunoprecipitation of sGC and LGN proteins fromthe lysate of BE2 human neuroblastoma cell line. Tran-sient overexpression of LGN in sGC expressingMDA468 breast cancer cell line markedly decreased theactivity of sGC in cell lysates in a gene-dose dependentfashion. On the contrary, inhibition of LGN expressionby targeted siRNA increased sGC activity in the cellularlysates. In vitro pull-down experiments performed withtruncated versions of LGN and sGC mapped thedomains required for interaction between LGN andsGC. However, when the effect of purified LGN proteinon the activity of purified sGC was tested in vitro, wefound no evidence of inhibition, suggesting that LGN

does not directly affect sGC function. When the sameexperiment was performed in the lysate of COS7 cell,we observed a marked inhibition of sGC.

ConclusionThese data suggest that LGN may function as an adap-tor protein for additional cellular factors inhibiting sGC.Since LGN is a known regulator of G-protein, whichassociates with Ga subunit, it may function as an adap-tor protein for a complex consisting of sGC, LGN andGa, thus connecting the NO-dependent and G-proteinsignaling pathways.

Published: 1 August 2011

doi:10.1186/1471-2210-11-S1-P15Cite this article as: Chauhan et al.: G-protein regulator LGN inhibits theactivity of soluble guanylyl cyclase. BMC Pharmacology 2011 11(Suppl 1):P15.

Submit your next manuscript to BioMed Centraland take full advantage of:

• Convenient online submission

• Thorough peer review

• No space constraints or color figure charges

• Immediate publication on acceptance

• Inclusion in PubMed, CAS, Scopus and Google Scholar

• Research which is freely available for redistribution

Submit your manuscript at www.biomedcentral.com/submit

* Correspondence: [email protected] of Texas Houston Medical School, Department of InternalMedicine, Division of Cardiology, Houston, Texas, USA

Chauhan et al. BMC Pharmacology 2011, 11(Suppl 1):P15http://www.biomedcentral.com/1471-2210/11/S1/P15

© 2011 Chauhan et al; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.

mailto:[email protected]

http://creativecommons.org/licenses/by/2.0