Selective expression of subtypes of receptors in mammalian cell lines
permits the study of the regulation of receptors in a homogeneous popu
lation of cells growing under controlled conditions. cDNAs encoding th
e human D2L and D2S receptors were ligated into a eukaryotic expressio
n vector, pRc/CMV. The resulting plasmid, which contains a cytomegalov
irus promoter for high expression levels, was used for stable transfec
tion of 293 cells, a human kidney cell line. Expression of D2L and D2S
receptors in 293 cells was confirmed by radioligand binding assays wi
th [I-125]NCQ 298. The pharmacological properties of the expressed rec
eptors were comparable to those of receptors in rat striatal homogenat
es and in other transfected cell lines. D2L and D2S receptors were cou
pled to inhibition of cAMP accumulation in 293 cells. Incubation of 29
3-D2L cells with agonists resulted in an increase in the density of D2
receptors without a change in the affinity of the receptors for [I-12
5]NCQ 298. This effect was time dependent, with a t1/2 of approximatel
y 6 hr. The dose dependence of up-regulation followed the pharmacologi
cal profile expected of a D2 receptor, with an order of potency of NS-
propylnorapomorphine (NPA) > quinpirole > dopamine. The density of rec
eptors was further increased by incubation of cells with agonist toget
her 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 no
t 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 [I-125]iodocyanopindolol. Levels of b
eta-adrenergic receptors in transfected 293-beta2 or 293-D2L cells did
not increase after exposure to NPA but decreased after exposure to is
oproterenol. Cells expressing D2L receptors were incubated with antago
nists, including SCH-23390, sulpiride, haloperidol, clozapine, and epi
depride, alone or in combination with NPA. Incubation of cells with SC
H-23390 had no effect on the density of D2 receptors, and SCH-23390 di
d not block the effect of NPA. D2-selective antagonists increased the
density of receptors. D2L receptor mRNA levels were unchanged during a
gonist treatment. This suggests a role for translational or post-trans
lational mechanisms in the regulation of D2 receptor levels in transfe
cted cell lines.