Modulation of dopamine D1 receptor signaling by adenosine A1 receptors in Sf9 cells requires expression of Gi proteins

There are several evidences that some functions of D1 dopamine receptors can be modulated by colocalized adenosine A1 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 D1 and A1 receptors was confirmed by proper binding of specific radioligands [3H]SCH23390 (Kd = 1.1 ± 0.1 nM, Bmax = 2.2 ± 0.1 pmol/mg protein) and [3H]DPCPX (Kd = 2.1 ± 0.8 nM, Bmax = 2.9 ± 0.4 pmol/mg protein), respectively. The kinetics of [3H]SCH23390 binding corresponded to the simplest reversible bimolecular binding reaction of complex formation, with kon = 0.20 ± 0.02 min−1 nM−1 and koff = 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 A1 receptor to Gi proteins was confirmed by both GTPγS dependent agonist binding and inhibition of cAMP accumulation by N-cyclopentyladenosine (NCPA). Activation of the A1 receptor by NCPA had no significant influence on neither affinities of dopaminergic ligands nor the radioligand binding kinetics to the co-exprssed D1 receptors in Sf9 cell membranes. On the other hand, the activation of the A1 receptors inhibited the D1 receptor-specific accumulation of cAMP, but only in cells where Gi proteins were expressed with the receptors.