Characterization of [ [formula omitted]]5-hydroxytryptamine and [ [formula omitted]]spiperone binding sites in clathrin-coated vesicles from bovine brain

Abstract

Coated vesicles prepared from bovine brain cerebral cortex exhibited [ 3 H ]5-hydroxytryptamine (5-HT, serotonin) and [ 3 H ]spiperone binding activities. The binding activities were localized in the inner core vesicles. Binding reached an equilibrium level by 30–45 min at 30°C, and was reversed by the addition of 100 μM 5-HT for [ 3 H ]5-HT binding or 10 μM ketanserin for [ 3 H ]spiperone binding. The saturation binding experiments indicated a single class of binding sites for [ 3 H ]5-HT and [ 3 H ]spiperone with apparent K d values of 2.4 and 1.75 nM, respectively. The binding of [ 3 H ]5-HT was displaced by 5-HT and 8-hydroxy-2-(di- n-propylamino)-tetralin (8-OH-DPAT), but not by ketanserin. The binding of [ 3 H ]spiperone was displaced by spiperone and ketanserin but not by 5-HT or 8-OH-DPAT even at 1 mM. The coated vesicles were shown by immunoblotting assay to contain α-subunits of GTP-binding proteins, Gαs, Gαi2, Gαi3, Gαo and Gαq/11. Forskolin-stimulated adenylate cyclase activity in the coated vesicles was inhibited to 80% of the control level by 5-HT or 8-OH-DPAT. These results suggested that 5-HT 1A and 5-HT 2A receptors are present in bovine brain coated vesicles and that the 5-HT 1A receptors are coupled to adenylate cyclase activity via GTP binding proteins.