Using bimolecular fluorescence complementation to monitor the diffusion of adenosine-A1 and -A2A receptor dimers in the membranes of single cells

S.J. Briddon , F. Ciruela†, J. Gandia† & S.J. Hill. Institute of Cell Signalling, University of Nottingham, Nottingham, NG7 2UH. †Department of Biochemistry and Molecular Biology, University of Barcelona, 08028 Barcelona, Spain.

The A1- and A2A-adenosine receptors (A1-AR and A2A-AR respectively) can exist in the cell membrane as homo- and hetero-dimers (Ciruela et al., 2006). Bimolecular fluorescence complementation (BiFC, Hu et al., 2002) is designed to detect the interaction between two proteins which have been fused to either the N- or C-terminal fragments of yellow fluorescent protein (N-YFP or C-YFP). Following interaction a fluorescent full length YFP (wtYFP) is reconstituted. We have used BiFC in conjunction with fluorescence correlation spectroscopy (FCS) to specifically monitor the diffusion of A1-AR and A2A-AR dimers in microdomains of Chinese Hamster Ovary (CHO) cells.
Using standard molecular biology techniques, cDNA encoding the human A1-AR or A2A-AR was cloned into pcDNA3.1 to produce C-terminal fusions with wtYFP, C-YFP or N-YFP. CHO-K1 cells were transfected for 24h with a total of 0.15μg/well of DNA using Lipofectamine (Invitrogen) according to manufacturer’s instructions. After a further 24h incubation (30ºC/5%CO2), FCS measurements were carried out on the upper cell membrane and analysed as previously described (Briddon et al., 2004).

Table 1 . Diffusion times ( τD1/2 ) obtained from FCS readings. Data are shown as mean±s.e. mean for ‘n’ different cells. †P<0.05 and *P<0.01 vs. either wtYFP construct. (one way ANOVA, post-hoc Newman-Keuls test).

FCS measurements showed two diffusion components (τD1 and τD2, Table 1). As previously described, τD1 , represents a photophysical property of YFP (e.g. blinking), whilst τD2 represents translational diffusion. Homodimeric versions of the A1-AR and A2A-AR showed significantly faster diffusion than those fused to wtYFP.
These data show that BiFC in combination with FCS can be used to study, in isolation, the diffusional characteristics of A1-AR and A2A-AR homo- and hetero-dimers. Our observations also suggest that complemented dimeric species represent a faster diffusing fraction of the total receptor population. It remains to be established what factors contribute to the slow diffusion of the remainder of the non-complemented receptor species.

Hu, C.-D. et al. (2002) Mol.Cell, 9, 789-798.
Briddon, S.J. et al. (2004) Proc. Nat. Acad. Sci, ( USA), 101, 4673-4678.
Ciruela, F. et al. (2006) J.Neurosci, 26, 2080-2087.

Funded by the BBSRC and SAF2005-00903 from the Ministerio de Educación y Ciencia.