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Exploration of cyclic nucleotide guanosine 3 5 monophosphate (cGMP) signalling dynamics in vivo

Reference	BB/C000633/1
Principal Investigator / Supervisor	Professor Shireen Davies
Co-Investigators / Co-Supervisors	Professor Julian Dow
Institution	University of Glasgow
Department	Institute of Biomedical & Life Sciences
Funding type	Research
Value (£)	330,094
Status	Completed
Type	Research Grant
Start date	01/10/2004
End date	31/03/2008
Duration	42 months

Abstract

The role of cyclic nucleotides, especially cGMP, is crucial in physiological function but is underexplored in an organotypic context. Our previous work in a genetic model of transporting epithelia, the Drosophila tubule, has shown that the cellular localisation of cGMP is as important as cellular concentration in eliciting physiological response. Furthermore cGMP effector proteins (cG-kinases, cG-phosphodiesterases (PDEs), cG channels) and the enzymes which generate cGMP (guanylate cyclases), are localised to the same compartments of the cell/tissue. We intend to investigate cGMP localisation and gradients by use of a FRET reporter for cGMP, based on the catalytic domain of vertebrate cG-kinase (cygnet). We will express this (subcloned into pUASt) in defined sub-sets of tubule cells under GAL4 control, and make the very first in vivo measurements for cGMP in differentiated tissue. We will measure cGMP gradients under different conditions of cGMP stimulation (using neurohormones and nitric oxide donors, for example) which will allow us to compare cGMP signatures in defined cell-types, providing the first description of cGMP localisation and distribution in an organotypic context. The cGMP effector proteins cG-kinases and cG-PDEs, act as cellular sinks for cGMP; in order to delineate the role of cG-kinases, and cG-PDEs in cGMP gradients, we will overexpress genes encoding these proteins in the cygnet backgrounds; and then measure FRET intensity. Furthermore, as our work has shown that vertebrate and Drosophila cGMP effector proteins are targeted to the same cellular locations, we will assess the possibility of signalling complexes for cGMP by immunoprecipitation and mass spec analysis of the proteins therein. (Our preliminary date shows that it is possible to successfully perform pull-downs using cG-PDE antibodies from Drosophila tissues.) This will allow the determination of (novel) components of cGMP effector complexes. Co-expression studies between putative components and cG-PDEs and cG-kinases will be performed to verify the pull-down data. These data will be correlated with the data from cygnet experiments.

Summary

unavailable

Committee	Closed Committee - Animal Sciences (AS)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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