The human adenosine A₁ receptor (A₁R) is a useful model of GPCR binding and activation. We have reported that the dissociation kinetics of a [ ³H]agonist from A₁R-G protein complexes are enhanced if an non-radioactive agonist rather than an antagonist/inverse agonist is used to prevent rebinding of the [ ³H]agonist (Hern et al., 2004a). This implies a novel mechanism by which binding information is transferred between high affinity binding sites on A₁R-G protein complexes. This could occur through a direct association of receptors as, for example, dimers. The aim of this study is to examine, using A₁R-GFP and A₁R-GFP-G _i fusion constructs, whether the ligand-selective induced [ ³H]agonist dissociation is sensitive to the receptor expression level.

A CHO cell line expressing high levels (HE) of the A₁R (ca 9 pmoles/mg protein) has been described previously (Hern et al., 2004a). Cell lines expressing either A₁R-GFP (10 lines) and A₁R-GFPG _i fusion constructs (23 lines) in CHO cells at different levels are described in the accompanying abstract (Hern et al., 2004b). Radioligand binding studies were carried out at 23^O in a Na/Mg/Hepes buffer (Browning et al, 2000). Membranes, pre-labelled with the agonist [ ³H]CHA ([ ³H]-N⁶-cyclohexyladenosine, 5 nM) for 60 min, were diluted 20 fold into buffer containing either non-radioactive CHA (1 µM) or DPCPX (1 µM) as a ‘chase’ ligand. Dissociation was allowed to progress for 10, 60 or 180 min, at which times the membranes were harvested by rapid filtration.

In all experiments, [ ³H]CHA dissociation was not monoexponential. For HE the % dissociation of [ ³H]CHA, with DPCPX as chase ligand, at 10, 60 and 180 min was 12 ± 1, 35 ± 1, and 52 ± 1%, respectively, n = 28. At these time points, the % dissociation of [ ³H]CHA in the presence of a CHA chase was greater, relative to the DPCPX chase, by 21 ± 1, 21 ± 1, and 13 ± 1%, respectively, n = 26-28. The values for % dissociation of [ ³H]CHA from A₁R-GFP- and A 1R-GFP-G _i-G protein complexes, in the presence of a DPCPX chase, at the three times (24 ± 1, 49 ± 1, 62 ± 1%, n = 13 [A₁R-GFP] and 19 ± 1, 45 ± 1, 62 ± 1, n = 30 [A₁R-GFP-G _i] respectively) were similar to each other and to A₁R (HE) and were independent of receptor expression level. In contrast, the CHA-enhanced [ ³H]CHA dissociation at the three times (15 ± 2, 16 ± 1, 10 ± 1%, n = 9-10), from both fusion constructs in higher expressing cell lines (9 lines, 7-13 pmoles/mg protein), was attenuated in lower expressing cell lines (4 ± 1, 8 ± 2, 4 ± 1%, n = 7-8 for 5 lines expressing 1-3 pmoles/mg); the individual values for the 33 cell lines examined progressively extrapolated towards 0 at zero receptor expression level.

The fact that [³H]CHA dissociation in the presence of CHA, but not in the presence of DPCPX, is sensitive to receptor expression levels of the two A₁R-GFP fusion constructs, suggests that agonist-induced agonist dissociation from these A₁R-G protein complexes is a manifestation of receptor-receptor interactions. The dependence of this interaction on receptor expression level, at least for A₁ adenosine receptors, is compatible with the coexistence of receptor monomers with dimers/oligomers.

Browning C et al., (2000) Br J Pharmacol 129, 42P
Hern J et al., (2004a) Br J Pharmacol in press
Hern J et al., (2004b) accompanying abstract