Single-Molecule Study Of The Oligomeric States Of The M2 Muscarinic Receptor, The Gi1 Protein And The M2-Gi1 Complex

Inconsistent results obtained in the identification of oligomers of rhodopsin-like GPCRs by various means and in various preparations has generated much debate on the role of oligomers. Estimates of size has largely focused on oligomers of the receptor yet it is the size of the receptor coupled to the G protein that is the most relevant but remains unknown. Using the photo-bleaching of fusions of single-molecules of eGFP, we evaluated the oligomeric size of the muscarinic M2 receptor, the Gi1 protein and various multimeric controls. The relationship between the number of photo-bleaching steps within homogeneous population of oligomeric controls of known size gave a distribution of bleaching steps instead of discrete steps. Statistical analysis of the distribution in terms of the binomial model yielded estimates of the oligomeric size. Using this approach, we examined the oligomeric size of both the receptor and the G protein in extracts that represent different stages of the signaling cycle: in the basal state, following activation by ligands, during and after coupling of the complex. The data indicate that whereas the receptor remains as a tetramer throughout the cycle, the oligomeric size of the G protein is dynamic. G proteins exist as hexamers in the basal state, couple as tetramers and uncouple from the receptor (i.e., fully activated) as dimers. Measurements with a receptor mimic to the wild-type G protein and a constitutively active mutant also yielded dimers. The spatial feasibility of a super-complex for the di-tetrameric arrangement of the receptor and G protein is presented using experimentally derived constraints.