A2a/D2 receptor interactions are not observed in COS-7 cells transiently transfected with dopamine D2 and adenosine A2a receptor cDNA.
The rat D2 receptor and the dog A2a receptor subcloned into the pXM vector were transiently transfected into COS-7 cells using the DEAE-dextran method. The transfected cells expressed approx. 200 fmol D2 receptors/mg protein and approx. 5 pmol/mg protein of the A2a receptor as judged by binding experiments with [3H]raclopride [or[3H]-N-propyl-apomorphine (NPA)] and [3H]-CGS 21680, respectively. The high affinity KD values were 0.43 and 19 nM for D2 and A2a receptors, respectively, in agreement with results obtained from other cells and tissues. The non-selective adenosine receptor agonist NECA stimulated cAMP accumulation both in non-transfected and transfected COS-7 cells with only a slight difference in potency, suggesting that most of the stimulation is due to activation of A2b receptors known to be present on virtually every cell. The two A2a selective agonists CGS 21680 and CV-1808 were essentially inactive in transfected COS-7 cells, but were very active in PC-12 cells known to possess functional A2a receptors. Dopamine did not decrease cAMP accumulation in the transfected COS-7 cells. CGS 21680 (30 nM) did not affect the binding characteristics of D2 receptors in the co-transfected COS-7 cells in contrast to the increased KH, KL and RH values found previously in rat striatal membranes after CGS 21680 treatment. The present findings indicate that transiently transfected A2a and D2 receptors in COS-7 cells have normal binding properties, but couple poorly to adenylyl cyclase, despite the presence of Gs protein and adenylyl cyclase in these cells. Our results also demonstrate that the previously reported interactions between A2a receptors and D2 receptors do not occur when only the receptor proteins are expressed in COS-7 cells, suggesting that the two receptor molecules do not interact directly to influence binding characteristics.
Snaprud
P
P
Department of Neuroscience, Karolinska Institute, Stockholm, Sweden.
Gerwins
P
P
Caron
M G
MG
Libert
F
F
Persson
H
H
Fredholm
B B
BB
Fuxe
K
K
D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D019830:Adenosine-5'-(N-ethylcarboxamide); D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D018076:DNA, Complementary / Q000737:chemistry; D016716:PC12 Cells; D010627:Phenethylamines / Q000494:pharmacology; D020891:Raclopride; D051381:Rats; D017448:Receptors, Dopamine D2 / Q000737:chemistry / Q000235:genetics*; D018047:Receptors, Purinergic P1 / Q000737:chemistry / Q000235:genetics*; D012457:Salicylamides / Q000494:pharmacology; D014162:Transfection
D018076:DNA, Complementary; D010627:Phenethylamines; D017448:Receptors, Dopamine D2; D018047:Receptors, Purinergic P1; D012457:Salicylamides; C061282:2-(4-(2-carboxyethyl)phenethylamino)-5'-N-ethylcarboxamidoadenosine; D019830:Adenosine-5'-(N-ethylcarboxamide); D020891:Raclopride; D000241:Adenosine