DRUG-INDUCED UP-REGULATION OF DOPAMINE D2 RECEPTORS ON CULTURED-CELLS

Ligand-induced up-regulation of recombinant dopamine D2 receptors was assessed using C-6 glioma cells stably expressing the short (415-amino -acid; D2(S)) and long (444-amino-acid; D2(L)) forms of the receptor, Overnight treatment of C6-D2(L) cells with N-propylnorapomorphine (NPA ) caused a time- and concentration-dependent increase in the density o f receptors, as assessed by the binding of radioligand to membranes pr epared from the cells, with no change in the affinity of the receptors for the radioligand. The effect of 10 mu M NPA was maximal after 10 h , at which time the density of D2(L) receptors was more than doubled. The agonists dopamine and quinpirole also increased the density of D2( L) receptors. The receptor up-regulation was not specific for agonists , because the antagonists epidepride, sulpiride, and domperidone cause d smaller (30-60%) increases in receptor density. Prolonged treatment with 10 mu M NPA desensitized D2(L) receptors, as evidenced by a reduc ed ability of dopamine to inhibit adenylyl cyclase, whereas treatment with sulpiride was associated with an enhanced responsiveness to dopam ine. The magnitude of NPA-induced receptor up-regulation in each of fo ur clonal lines of C6-D2(L) (mean increase, 80%) was greater than in a ll four lines of C6-D2(S) cells (33%). Inactivation of pertussis toxin -sensitive G proteins had no effect on the basal density of D2(L) rece ptors or on the NPA-induced receptor up-regulation. Treatment with 5 m u g/ml of cycloheximide, on the other hand, decreased the basal densit y of receptors and attenuated, but did not prevent, the NPA-induced in crease. Chimeric D1/D2 receptors were used to identify structural dete rminants of dopamine receptor regulation. Treatment with the D1/D2 ago nist NPA decreased the density of D1 and chimeric CH4 and CH3 receptor s. The latter two receptors have D1 sequence from the amino-terminus t o the amino-terminal end of transmembrane region (TM) VII and VI, resp ectively. CH2, with D1 sequence up to the amino-terminal end of TM V, and thus the third cytoplasmic loop of the D2 receptor, was up-regulat ed by NPA or the D2-selective agonist quinpirole. Quinpirole treatment decreased the density of CH3 acid had no effect on CH4 or D1 receptor s. The different responses of CH2 and CH3 to agonist treatment suggest a role for TM V and the third cytoplasmic loop in the direction of re ceptor regulation.