Classically, activated seven helical receptors lead to G-protein heterotrimer dissociation into Gα-GTP and free Gβγ.Both these signalling species can then interact with their effectors. The signal is terminated by the intrinsic hydrolysis rate of the G-protein alpha subunit, which may be further accelerated by regulators of G-protein signalling (RGS). We have generated and characterised cyan (CFP) and yellow fluorescent (YFP) derivatives of specific examples of these proteins (GoαA-YFP, GoαA-CFP, Gγ2-CFP, RGS8-YFP, adenosine receptor: A1-CFP, dopamine receptor: D2SCFP. These proteins were functional when assayed with patch clamping after transient transfection into HEK293 cells expressing a G-protein gated K channel with or without various G-protein coupled receptors as appropriate. We next used fluorescent microscopy with either a laser scanning confocal microscope or CCD based system to explore protein-protein interactions in HEK293 cells between these components using fluorescence resonance energy transfer (FRET, FRET ratios (FR) measured using the three-cube technique, mean±S.E.M.). RGS-YFP formed a stable interaction with GoαA-CFP that was not modulated significantly by agonist application e.g. confocal: FR=2.06±0.1 (n=11), CCD: FR=2.73±0.3 (n=6). The interaction between the two proteins was lost with an N-terminally deleted RGS8-YFP (CCD: FR=1.18±0.03, n=6) and partially with a RGS-insensitive mutant of GoαA (CCD: FR=1.65±0.27, n=9). In contrast, RGS-YFP did not interact with A1-CFP (confocal: FR=1.04±0.05, n=13) or with D2-CFP (confocal: FR=1.04±0.05, n=22). Finally, we observed FRET, restricted to the plasma membrane, between the α and γ subunits of the G-protein showing interaction between these subunits (CCD: FR=1.54±0.13, A1 cell line, n=10; FR=1.97±0.28, M4 cell line, n=9). This interaction was partially lost after relevant agonist application in the A1 (FR=1.30±0.12) and M4 (FR=1.5±0.22) cell lines. In conclusion, our studies reveal a network of scaffolding interactions between relevant proteins in this signalling system. We are beginning to explore the functional consequences for signal transduction and develop methods to improve temporal and spatial resolution.
University of Glasgow (2004) J Physiol 557P, PC90
Communications: Examining the interaction of G-proteins, seven helical receptors and regulators of G-protein signalling using fluorescence resonance energy transfer.
M. Nobles (a),A.Benians (b) and A. Tinker ©
(a) Department of Medicine, Centre for Clinical Pharmacology, University College London, 5 University Street, London WC1E 6JJ, UK, (b) Department of Medicine, Centre for Clinical Pharmacology, University College London, 5 University Street, London WC1E 6JJ, UK and (c) Department of Medicine, Centre for Clinical Pharmacology, University College London, 5 University Street, London WC1E 6JJ, UK
View other abstracts by:
Where applicable, experiments conform with Society ethical requirements.