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

PURPOSE To examine the interaction of alpha 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-Gi alpha), 3646 (anti-Gi alpha 1), 1521 (anti-Gi alpha 2), and 1518 (anti-Gi alpha 3), 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 alpha subunits indicate that the A1 adenosine receptors from these cells are specifically coupled to Gi alpha complexes. The results further indicate that the A1 adenosine receptors are predominantly associated to Gi alpha-3. CONCLUSION The findings document a selective interaction between the alpha subunits of the inhibitory guanine nucleotide binding protein (Gi) and A1 adenosine receptors in ocular ciliary epithelial cells in culture. The results suggest that adenosine receptors coupled to Gi alpha-3 may provide a site at which modulation of aqueous humor production in the ciliary epithelium occurs via the G protein-adenylyl cyclase pathway.