Speaker
Mr
Dennis D. Fernandes
(Department of Physics, University of Toronto)
Description
The role of oligomers in signalling via G protein-coupled receptors (GPCRs) has been under debate. The oligomeric size of several GPCRs has been studied using fluorescence-based techniques and have provided inconsistent results, while their attendant G proteins have received little attention. In this study, the oligomeric nature of the GPCR signalling complex comprising the M2 muscarinic receptor (M2R) and the heterotrimeric G protein Gi1 (Gαi1β1γ2) has been probed using stepwise fluorescence photobleaching. A hexahistidyl-tag and a fluorophore (eGFP) were fused at the N- or C-terminus of M2R, and inserted into Gαi1 (at position 91) or Gγ2 without perturbing the nucleotide-binding affinity of Gαi1 or the interaction with M2R and the Gβ1γ2 heterodimer. Complexes of receptors and G proteins were immobilised on histidine-specific surfaces and their oligomeric size was derived by analyzing the distribution of stepwise changes in fluorescence intensity.
Immobilised eGFP-M2R and eGFP- Gαi1β1γ2 both showed four photobleaching steps, which implies a tetramer, and both remain as tetramers when co-expressed and co-purified as the signalling complex. The agonist carbachol, the antagonist N-methylscopolamine (NMS) and the nucleotide guanosine triphosphate (GTP), only affected the oligomeric composition and integrity of either protein when coupled to the other. These findings were corroborated with pair-wise FRET efficiencies measured at the membrane of living cells. Upon activation via M2R, the number of photobleaching steps for Gαi1β1γ2 was reduced to 1-2 after the addition of GTP, suggesting disaggregation into monomers or dimers, and had no effect on M2R. These results were supported with FRET and molecular dynamic simulations, which suggests that G proteins undergo conformational changes upon binding of M2R and GTP. The concerted action of these conformational changes induces movement or alignment of alpha-helical domains which results in the proliferation or disruption of the oligomeric interface between the receptor and the G protein.
Primary authors
Mr
Dennis D. Fernandes
(Department of Physics, University of Toronto)
Ms
Huiqiao Ji
(Department of Pharmaceutical Sciences, University of Toronto)
Dr
Rabindra V. Shivnaraine
(Department of Pharmaceutical Sciences, University of Toronto)
Co-authors
Mr
Brendan Kelly
(Department of Pharmaceutical Sciences, University of Toronto)
Dr
Claudiu C. Gradinaru
(Department of Physics, University of Toronto)
Dr
Fei Huang
(Department of Pharmaceutical Sciences, University of Toronto)
Dr
James W. Wells
(Department of Pharmaceutical Sciences, University of Toronto)
Mr
Jonathon V. Rocheleau
(Institute of Biomedical and Biomaterial Engineering, University of Toronto.)
Ms
Krishana S. Sankar
(Department of Physiology, University of Toronto)
Mr
Yuchong Li
(Department of Physics, University of Toronto)
Mr
Zhenfu Zhang
(Department of Physcis, University of Toronto)
