Immunoprecipitation of A₁ adenosine receptor-GTP-binding protein complexes in ciliary epithelial cells

Purpose. To examine the interaction of α subunits of guanine nucleotide binding proteins with A₁ adenosine receptors from the SV40-transformed bovine-derived pigmented (PE) and human-derived nonpigmented (NPE) ciliary epithelial cell lines using an immunoprecipitation approach and [³H]DPCPX, a selective radioligand to adenosine receptor. Methods. Solubilized preparations of adenosine receptors from PE and NPE cell lines were immunoprecipitated with G protein specific antisera 8730 (anti-G(iα)), 3646 (anti-G(iα1)), 1521 (anti-G(iα2)), and 1518 (anti-G(iα3)), and of adenosine receptor-G protein complexes were detected by the binding of radioactive [³H]DPCPX. Results. Data indicate that [³H]DPCPX forms high- affinity complex with membrane-bound and solubilized forms of adenosine receptors from PE and NPE cells. Peptide-directed antisera against various G protein α subunits indicate that the A₁ adenosine receptors from these cells are specifically coupled to G(iα) complexes. The results further indicate that the A₁ adenosine receptors are predominantly associated to G(iα-3). Conclusion. The findings document a selective interaction between the α subunits of the inhibitory guanine nucleotide binding protein (G(i)) and A₁ adenosine receptors in ocular ciliary epithelial cells in culture. The results suggest that adenosine receptors coupled to G(iα-3) may provide a site at which modulation of aqueous humor production in the ciliary epithelium occurs via the G protein-adenylyl cyclase pathway.