SEQUESTRATION OF THE SHORT AND LONG ISOFORMS OF DOPAMINE D-2 RECEPTORS EXPRESSED IN CHINESE-HAMSTER OVARY CELLS

The short (D-2S) and long (D-2L) isoforms of dopamine D-2 receptors we re stably expressed in Chinese hamster ovary cells, and dopamine-induc ed sequestration was examined by measuring the loss of binding of the hydrophilic ligand [H-3]sulpiride from the cell surface. Dopamine trea tment of Chinese hamster ovary cells expressing D-2S for 30 min at 37 degrees caused a 43.8 +/- 3.4% decrease in [H-3]sulpiride binding acti vity measured by incubation of the treated cells with [H-3]sulpiride a t 4 degrees for 4 hr after the dopamine was washed out. The half-life of the decrease in binding was estimated to be 18.7 +/- 1.6 min, and t he concentration of dopamine giving a half-maximal effect (EC(50)) was estimated to be 180 +/- 90 nM. The decrease was reversible, and the b inding activity was recovered by washing out the dopamine and incubati ng the cells at 37 degrees for 30 min but was not reversible when the cells were incubated at 4 degrees. The binding activity of [H-3]spiper one, a hydrophobic ligand, was not affected by the dopamine treatment under the same experimental conditions. These results indicate that ap proximately one half of the D-2S receptors undergo agonist-induced seq uestration, probably endocytosis, in a reversible and temperature-depe ndent manner. Sequestration of D-2L receptors was not as apparent as t hat of D-2S receptors; the decrease in [H-3]sulpiride binding activity was 21.6 +/- 0.9% and the rate of the decrease was delayed, with a ha lf-life of 33.2 +/- 7.8 min, although effective concentrations of dopa mine were similar, with EC(50) = 170 +/- 50 nM. A D-2S receptor varian t containing a missense mutation changing Ser(311) in the third intrac ellular loop to cysteine was found to be sequestered to a significantl y lesser extent than with wild-type D-2S receptors. This finding was d iscussed with respect to the report that this variant gene is found mo re frequently in schizophrenic patients than in control subjects.