Modulation of dopamine D 1 receptor signaling by adenosine A 1 receptors in Sf9 cells requires expression of G i proteins

Abstract

There are several evidences that some functions of D 1 dopamine receptors can be modulated by colocalized adenosine A 1 receptors. To elucidate the role of particular components of the receptor complex in the ligand binding and second messenger activation level we have used Sf9 cell expression system. The expression of D 1 and A 1 receptors was confirmed by proper binding of specific radioligands [ 3 H]SCH23390 ( K d = 1.1 ± 0.1 nM, B max = 2.2 ± 0.1 pmol/mg protein) and [ 3 H]DPCPX ( K d = 2.1 ± 0.8 nM, B max = 2.9 ± 0.4 pmol/mg protein), respectively. The kinetics of [ 3 H]SCH23390 binding corresponded to the simplest reversible bimolecular binding reaction of complex formation, with k on = 0.20 ± 0.02 min −1 nM −1 and k off = 0.13 ± 0.01 min −1. Dopaminergic agonists increased the accumulation of cAMP in the transfected cells in concentration-dependent manner, indicating a correct coupling of receptor to second messenger system. The coupling of the A 1 receptor to G i proteins was confirmed by both GTPγS dependent agonist binding and inhibition of cAMP accumulation by N-cyclopentyladenosine (NCPA). Activation of the A 1 receptor by NCPA had no significant influence on neither affinities of dopaminergic ligands nor the radioligand binding kinetics to the co-exprssed D 1 receptors in Sf9 cell membranes. On the other hand, the activation of the A 1 receptors inhibited the D 1 receptor-specific accumulation of cAMP, but only in cells where G i proteins were expressed with the receptors.