Activation of the G-protein coupled receptor (GPCR) chemokine receptor 5 (CCR5) has been shown to result in activation of the Gαi family of heterotrimeric G-proteins. However, little is currently known about the temporal characteristics of G-protein heterotrimer activation by CCR5. Furthermore, this simplistic model does not account for the range of changes CCR5 activation brings about. Recent evidence suggests that GPCRs may be able to signal through numerous permutations of heterotrimer. In order to assess G-protein activation and interaction with CCR5 functional stably transfected cell lines were created expressing Gαi2 fused to ECFP and Gβ1 fused to EYFP. The interaction of these constructs was confirmed by measuring fluorescent resonance energy transfer (FRET). Stably transfected cells exhibited a FRET ratio of 2.63% (±0.345%, n=3) over that of control cells. Following CCR5 stimulation with CCL3, Gαi2β1γ activation could be monitored in real time, in whole cell populations, on a fluorescent plate reader by monitoring FRET emissions. This assay system represents a novel approach to measuring G-protein activation which can be used as a foundation to build more powerful FRET based assays. Measurement of G-protein interactions was further invested by BRET based studies. Transfection of dominant negative and constitutively active G-proteins alongside siRNA knockdown of G-proteins revealed that CCR5 is capable of signalling through other members of the Gαi family, with strikingly similar efficacy and potency to CCL3 stimulation. Dual knockdown of Gαi subunits and Gαq resulted in much attenuated calcium release following CCL3 stimulation, providing evidence that CCR5 may functionally couple to several types of G-proteins. Findings also support the theory that GPCRs can participate in domain swapping in order to rescue function. Treatment with gallein resulted in higher resting cytosolic cAMP but did not prevent CCR5 mediated inhibition of cAMP production. Gallein treatment also resulted in significant increases in calcium release following CCR5 activation, highlighting Gβγ as a potential target for modulating CCR5 signalling events. Data herein emphasize the complexity of GPCR signalling, and also provide a foundation for exploring GPCR signalling using fluorescence resonance energy transfer methods in the future.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:569309 |
| Date | January 2010 |
| Creators | Kerr, Jason Stuart |
| Publisher | University of East Anglia |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://ueaeprints.uea.ac.uk/10571/ |
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