Paradoxical regulation of dopamine receptors in transfected 293 cells.
Selective expression of subtypes of receptors in mammalian cell lines permits the study of the regulation of receptors in a homogeneous population of cells growing under controlled conditions. cDNAs encoding the human D2L and D2S receptors were ligated into a eukaryotic expression vector, pRc/CMV. The resulting plasmid, which contains a cytomegalovirus promoter for high expression levels, was used for stable transfection of 293 cells, a human kidney cell line. Expression of D2L and D2S receptors in 293 cells was confirmed by radioligand binding assays with [125I]NCQ 298. The pharmacological properties of the expressed receptors were comparable to those of receptors in rat striatal homogenates and in other transfected cell lines. D2L and D2S receptors were coupled to inhibition of cAMP accumulation in 293 cells. Incubation of 293-D2L cells with agonists resulted in an increase in the density of D2 receptors without a change in the affinity of the receptors for [125I]NCQ 298. This effect was time dependent, with a t1/2 of approximately 6 hr. The dose dependence of up-regulation followed the pharmacological profile expected of a D2 receptor, with an order of potency of N-propylnorapomorphine (NPA) > quinpirole > dopamine. The density of receptors was further increased by incubation of cells with agonist together with forskolin or 8-bromo-cAMP. D2S receptors responded similarly to D2L receptors to treatment with NPA and forskolin. Exposure of 293-D2L cells to the beta-adrenergic receptor agonist isoproterenol did not change the density of D2L receptors. Similarly, NPA had no effect on levels of endogenously expressed beta-adrenergic receptors in 293-D2L cells, as assayed by binding of [125I]iodocyanopindolol. Levels of beta-adrenergic receptors in transfected 293-beta 2 or 293-D2L cells did not increase after exposure to NPA but decreased after exposure to isoproterenol. Cells expressing D2L receptors were incubated with antagonists, including SCH-23390, sulpiride, haloperidol, clozapine, and epidepride, alone or in combination with NPA. Incubation of cells with SCH-23390 had no effect on the density of D2 receptors, and SCH-23390 did not block the effect of NPA. D2-selective antagonists increased the density of receptors. D2L receptor mRNA levels were unchanged during agonist treatment. This suggests a role for translational or post-translational mechanisms in the regulation of D2 receptor levels in transfected cell lines.
Filtz
T M
TM
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104-6084.
Artymyshyn
R P
RP
Guan
W
W
Molinoff
P B
PB
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D001058:Apomorphine / Q000031:analogs & derivatives / Q000494:pharmacology; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D003342:Corpus Striatum / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004298:Dopamine / Q000494:pharmacology; D015259:Dopamine Agents / Q000494:pharmacology; D018492:Dopamine Antagonists; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D011869:Radioligand Assay; D051381:Rats; D017448:Receptors, Dopamine D2 / Q000378:metabolism*; D014162:Transfection
D015259:Dopamine Agents; D018492:Dopamine Antagonists; D017448:Receptors, Dopamine D2; D005576:Colforsin; D015124:8-Bromo Cyclic Adenosine Monophosphate; C009074:N-n-propylnorapomorphine; D004247:DNA; D000242:Cyclic AMP; D007545:Isoproterenol; D001058:Apomorphine; D004298:Dopamine