THE D-2S AND D-2L DOPAMINE-RECEPTOR ISOFORMS ARE DIFFERENTIALLY REGULATED IN CHINESE-HAMSTER OVARY CELLS

To investigate and compare the regulatory properties of the two isofor ms of the D-2 dopamine receptor, we have stably expressed their cDNAs in Chinese hamster ovary (CHO) cells. Cell lines were selected that ex press similar levels of [H-3]methylspiperone-binding activity. Both is oforms mediate a dose-dependent and pharmacologically specific inhibit ion of adenylyl cyclase activity in both intact cell and membrane prep arations. Pretreatment of both D-2L and D-2S receptor-expressing cells with 100 mu M dopamine produces a similar to 5-fold shift (to lower a ffinity) in the EC(50) for dopamine inhibition of cAMP accumulation, w ith a 25-30% decrease in the maximum response. Dopamine treatment also results in a similar to 25% decrease in the maximum receptor binding activity of the D-2S receptor-expressing cells. In contrast, the D-2L receptors are up-regulated by about 2-fold in response to dopamine exp osure. This difference in response between the D-2S and D-2L receptors is not cell line specific, inasmuch as other CHO clones expressing th ese isoforms show identical responses. The dopamine-induced up-regulat ion of D-2L receptor binding is time dependent, reaching maximal level s after 10 hr (t(1/2) = 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 (t(1/2) = 5 hr) than the receptor up-regulation. B oth regulatory responses are induced in a dose-dependent fashion by do pamine, albeit with different potencies (up-regulation EC(50) = 100 nM , desensitization EC(50) = 2 mu M). These regulatory effects are pharm acologically specific, being mimicked by D-2-selective agonists but no t by agonists of other receptor subtypes. The dopamine-induced recepto r up-regulation is blocked by prior treatment of the cells with pertus sis toxin and is not mimicked by cAMP analogs. Conversely, elevation o f intracellular cAMP levels results in down-regulation of the D-2L rec eptor activity. To test whether protein synthesis is required for the D-2L receptor up-regulation, cycloheximide was used to block mRNA tran slation. This was found to completely inhibit the up-regulation of D-2 L binding activity; however, there was no effect on the desensitizatio n of the adenylyl cyclase response. RNA dot-blot analyses indicate tha t dopamine treatment is associated with a sustained 2-fold increase in the steady state levels of D-2L mRNA, whereas D-2S mRNA is transientl y increased by only 50%. Our data suggest that dopamine can promote a number of regulatory events in the CHO cells, including desensitizatio n of both D-2 receptor isoforms, down-regulation of the D-2S isoform, and upregulation of the D-2L receptor.