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+/-0.8nM, 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 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 A1 receptor to Gi proteins was confirmed by both GTPgammaS 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.
Uustare
Ain
A
Institute of Organic and Bioorganic Chemistry, University of Tartu, Tartu, Estonia.
Reinart
Reet
R
Rinken
Ago
A
D015647:2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine / Q000494:pharmacology; D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D058916:Adenosine A1 Receptor Antagonists; D000818:Animals; D001552:Benzazepines / Q000493:pharmacokinetics; D001667:Binding, Competitive / Q000187:drug effects; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D018491:Dopamine Agonists / Q000494:pharmacology; D018492:Dopamine Antagonists / Q000493:pharmacokinetics; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D011869:Radioligand Assay / Q000379:methods; D043682:Receptor, Adenosine A1 / Q000502:physiology*; D017447:Receptors, Dopamine D1 / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D018411:Spodoptera; D014162:Transfection / Q000379:methods; D014316:Tritium / Q000493:pharmacokinetics; D014970:Xanthines / Q000493:pharmacokinetics
D058916:Adenosine A1 Receptor Antagonists; D001552:Benzazepines; D018491:Dopamine Agonists; D018492:Dopamine Antagonists; D043682:Receptor, Adenosine A1; D017447:Receptors, Dopamine D1; D014970:Xanthines; D014316:Tritium; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D015647:2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; C051360:1,3-dipropyl-8-cyclopentylxanthine; D000242:Cyclic AMP; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D000241:Adenosine