Effect of phospholipases and proteases on the [3H]N6‐(R)‐phenylisopropyladenosine ([3H]R‐PIA) binding to A1 adenosine receptors from pig cerebral cortex

Abstract

The effect of phospholipases and proteases on the membrane-bound and solubilized A₁ adenosine receptor has been studied. Phospholipids modulate the [³H]N⁶-(R)-phenylisopropyladenosine binding to A₁ adenosine receptors in crude membranes and in soluble preparations, because changes in the phospholipid environment decrease both the binding capacity and the affinity for the ligand. It has become clear that (1) there is co-solubilization of receptor and phospholipids; (2) the phospholipid requirements are different for the coupled and the uncoupled receptor; (3) a net charge in the polar head produced by phospholipase D prevents the agonist binding to the receptor-G protein complex; alternatively, when the whole polar head is removed by phospholipase C the uncoupled receptor is altered; and (4) the protease action upon the receptor suggests that receptor coupled to G protein is more protected by the membrane than the uncoupled receptor. In kinetic experiments performed on membranes it was demonstrated that phospholipase C and trypsin increased the K_d value of the high-affinity state by modifying both k₁ and k₋₁. In contrast they only modified the dissociation constant of the low-affinity state. In conclusion it should be noted that phospholipids play a key role for the binding of R-PIA to A₁ adenosine receptor. Also, a different disposition within the membrane of the coupled and uncoupled receptor is encountered.