To investigate and compare the regulatory properties of the two isoforms of the D2 dopamine receptor, we have stably expressed their cDNAs in Chinese hamster ovary (CHO) cells. Cell lines were selected that express similar levels of [3H]methylspiperone-binding activity. Both isoforms mediate a dose-dependent and pharmacologically specific inhibition of adenylyl cyclase activity in both intact cell and membrane preparations. Pretreatment of both D2L and D2S receptor-expressing cells with 100 microM dopamine produces a approximately 5-fold shift (to lower affinity) in the EC50 for dopamine inhibition of cAMP accumulation, with a 25-30% decrease in the maximum response. Dopamine treatment also results in a approximately 25% decrease in the maximum receptor binding activity of the D2S receptor-expressing cells. In contrast, the D2L receptors are up-regulated by about 2-fold in response to dopamine exposure. This difference in response between the D2S and D2L receptors is not cell line specific, inasmuch as other CHO clones expressing these isoforms show identical responses. The dopamine-induced up-regulation of D2L receptor binding is time dependent, reaching maximal levels after 10 hr (t1/2 = 2 hr). Upon removal of dopamine, the receptor binding activity returns to control levels within 20 hr. The adenylyl cyclase desensitization response is also time dependent but exhibits a slower time course (t1/2 = 5 hr) than the receptor up-regulation. Both regulatory responses are induced in a dose-dependent fashion by dopamine, albeit with different potencies (up-regulation EC50 = 100 nM, desensitization EC50 = 2 microM). These regulatory effects are pharmacologically specific, being mimicked by D2-selective agonists but not agonists of other receptor subtypes. The dopamine-induced receptor up-regulation is blocked by prior treatment of the cells with pertussis toxin and is not mimicked by cAMP analogs. Conversely, elevation of intracellular cAMP levels results in down-regulation of the D2L receptor activity. To test whether protein synthesis is required for the D2L receptor up-regulation, cycloheximide was used to block mRNA translation. This was found to completely inhibit the up-regulation of D2L binding activity; however, there was no effect on the desensitization of the adenylyl cyclase response. RNA dot-blot analyses indicate that dopamine treatment is associated with a sustained 2-fold increase in the steady state levels of D2L mRNA, whereas D2S mRNA is transiently increased by only 50%.(ABSTRACT TRUNCATED AT 400 WORDS)