A guanosine 3',5'-cyclic monophosphate (cGMP) reporter system based on the G-kinase/CREB/CRE signal transduction pathway |
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Authors: | Okano Ichiro Miyazato Mikiya Kangawa Kenji |
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Institution: | Department of Biochemistry, National Cerebral and Cardiovascular Center Research Institute, Fujishiro-dai, Suita, Osaka, Japan |
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Abstract: | Guanylate cyclases constitute a gene family of enzymes that synthesize the second messenger guanosine 3′,5′-cyclic monophosphate (cGMP) and play important roles in diverse physiological functions. Here we report a novel, simple and highly sensitive method for measurement intracellular cGMP concentrations using a cAMP-responsive element (CRE) and cGMP-dependent protein kinase (cGK). Transient transfection of the CRE reporter plasmid, encoding a luciferase reporter gene under the control of a modified promoter containing a CRE, and a cGK expression vector into HEK293 cells followed by treatment with 8-bromo-cGMP showed a dose dependent increase in luciferase activity. Moreover, HEK293 cells expressing GC-A or GC-B natriuretic peptide receptors and harboring this reporter system responded to specific ligands in a dose dependent manner. Our results indicate that this reporter gene method enables high throughput screening of receptor-type GC selective agonists in the treatment of cardiovascular diseases and homeostatic dysfunctions. |
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Keywords: | Abbreviations: GC guanylate cyclase cGK cGMP-dependent protein kinase CRE cAMP-responsive element CREB CRE binding protein cAK cAMP-dependent protein kinase MMTV mouse mammary tumor virus |
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