An Investigation Into the Membrane Diffusion and Organisation of Adenosine Receptor Homo-oligomers

Abstract

Oligomerisation of G-protein coupled receptors (GPCRs) is now a widely accepted phenomenon, although its effects on receptor signalling, pharmacology and organisation are still unclear. Using a combination of bimolecular fluorescence complementation (BiFC, Hu, Mol. Cell, 9, 789, 2002) and fluorescence correlation spectroscopy (FCS), we have specifically monitored the diffusion of homo-oligomers of three members of the adenosine receptor family of GPCRs (the A1-, A2A- and A3-adenosine receptors (Ax-AR)) in microdomains of living cells. This approach has allowed us to directly investigate the membrane organisation of homo-oligomeric forms of these receptors.FCS measurements were carried out as previously described (Briddon, PNAS, 101, 4673, 2004).on the upper cell membrane of CHO-K1 cells transiently expressing C-terminal fusions of each AR subtype with either wtYFP (representing total receptor population) or C-YFP and N-YFP (representing oligomeric receptors).Homo-oligomers of all three subtypes were detected and showed a high degree of membrane localisation. For all three subtypes, receptors labelled with wtYFP (total receptor population) showed similar diffusion co-efficients (D=0.40, 0.51 and 0.43 μm2/s for A1-, A2A- and A3-AR, respectively). The oligomeric A3-AR (measured using BiFC) had a significantly faster diffusion co-efficient when compared to the A3-AR total population (D=0.60 vs. 0.43 μm2/s, P<0.05) suggesting that the homo-oligomeric A3-AR represents a faster diffusing fraction of the total receptor population. This was not the case for the A1- and A2A-ARs. Further investigation into the extent of receptor dimerisation for each ARs subtype among the total population and their membrane mobilities was carried out using photon counting histogram (PCH) analysis and fluorescence recovery after photobleaching (FRAP). These data indicate important differences in the molecular organisation of the monomeric vs oligomeric forms of the A3-AR, and also differences among receptor subtypes in their propensity for dimer formation.