Ligand binding inhibitors of A1 adenosine receptor from Rana rugosa are phospholipase A2s

Inhibitors of the A1 Adenosine Receptor were isolated from the skin extract of Korean frog, Rana rugosa. The frog-skin extract was prepared by an electrical shock and fractionated with C4 followed by C18 reverse-phase HPLC. Two A1 receptor inhibitors were isolated using a filter binding assay and the molecular masses of the proteins were estimated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry to be 15 347 and 15 404 Da, respectively. The inhibitory activity was also measured against other membrane receptors, such as the A2 Adenosine Receptor, Muscarinic Acetylcholine Receptor and capsaicin receptor. Ligand binding to the A2 and muscarinic receptors was also severely inhibited by these proteins. However, they did not inhibit the functional activation of the capsaicin receptor by its ligand, capsaicin, suggesting that inhibition of ligand-receptor binding occurs specifically. Their N-terminal sequences were determined by Edman degradation. Surprisingly, they showed sequence similarity to the secretory protein, Phospholipase A2 from various organisms. The Phospholipase A2 activity of both proteins was tested using Dole's assay technique. Both proteins showed Phospholipase A2 activity, and therefore, they were designated as PLA2-R1 and PLA2-R2, respectively. In addition, their ligand-binding inhibitory activity depended on their Phospholipase A2 activity. This is the first finding that the frog secretes a Phospholipase A2 similar to that of Snake Venoms, which posess inhibitory activity against the adenosine A1, adenosine A2 and muscarinic receptors.