The mobility of the human adenosine A1 receptor-GFP fusion protein is dependent on receptor density at the surface of living CHO cells

Jonathan Hern, Stephen Hill, Stephen Briddon

The University of Nottingham, Nottingham, UK

The human adenosine A1 receptor can couple to different G proteins in an agonist and receptor density-dependent manner (Cordeaux et al., 2000). Density-dependent receptor-receptor interactions have also been reported for the adenosine A1 receptor-GFP fusion protein (A1-GFP) in purified cell membranes (Hern et al., 2004). Using fluorescence correlation spectroscopy (FCS), the aim of the current study was to investigate whether changes in density of A1-GFP expression caused changes in the molecular organisation of the receptor within small locales at the surface of living cells.

Seven stable Chinese Hamster Ovary (CHO) cell lines expressing A1-GFP were chosen from previous studies and [3H]DPCPX saturation experiments performed as previously described (Hern & Birdsall, 2004). FCS measurements were carried out on both apical and basal cell surfaces and receptor diffusion co-efficient and particle number derived from autocorrelation analysis as previously described (Briddon et al., 2004).

[3H]DPCPX saturation binding experiments at purified membranes showed a three-fold range in Bmax (6-18 pmol/mg) and no significant variance in log Kd (mean -8.78 ± 0.03, n=28). At the basal cell surface the A1-GFP diffusion coefficient showed a significant correlation with the density of A1-GFP fluorescent particles derived from the autocorrelation curve amplitude (Figure 1).

Figure 1; A1-GFP diffusion coefficient and particle density at apical and basal cell surfaces for all seven cell lines. Each data point shown is mean ± s.e.m of 33-58 cells for each cell line. FCS measurements at basal cell surfaces showed a significant. Pearson correlation (P < 0.05, r2 = 0.65) between A1-GFP density and mobility.

These results show that not only is A1-GFP differentially organised at apical and basal cell surfaces (Hern et al., 2008, this meeting), the mobility of A1-GFP particles at the basal cell surface is decreased with increasing density of expression. We show that over a pharmacologically relevant range of receptor expression (Cordeaux et al., 2000) FCS identifies significant changes in the mobility and density of cell surface receptors within small regions of the surface of living cells.

This work was supported by the BBSRC grant number BBD5215811

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http://www.pA2online.org/Vol2Issue2abst005P.html

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