Abstract

We have examined the cellular processes underlying the desensitization of the 5-hydroxytryptamine (5-HT)_2A receptor induced by agonist or antagonist exposure. Treatment of C6 glioma cells with either 5-HT or the 5-HT_2A receptor antagonist ketanserin resulted in an attenuation in 5-HT_2A receptor function, specifically the accumulation of inositol phosphates stimulated by the partial agonist quipazine. 5-HT-induced desensitization of the 5-HT_2A receptor involved receptor internalization through a clathrin- and dynamin-dependent process because it was prevented by concanavalin A, monodansylcadaverine, and by expression of the dominant negative mutants β-arrestin (319–418) and dynamin K44A. Although short-term (i.e., 10 min) 5-HT and ketanserin exposure resulted in the same degree of desensitization, ketanserin-induced desensitization was not prevented by these agents and did not involve receptor internalization. In contrast, prolonged ketanserin exposure (i.e., 2 h) resulted in 5-HT_2A receptor internalization through a clathrin- and dynamin-dependent process, as was observed after agonist treatment. Inhibitors of protein kinase C or calcium-calmodulin kinase II did not attenuate or prevent 5-HT-induced desensitization of the receptor. 5-HT_2A receptor desensitization induced by 5-HT and prolonged ketanserin treatment, but not by short-term ketanserin treatment, was prevented by the expression of the dominant negative mutant of G protein-coupled receptor kinase (GRK)2, GRK2-K220R, and by an anti-GRK2/3 antibody. Our data indicate a dual mechanism of early and late desensitization by the antagonist ketanserin. Short-term ketanserin treatment reduced the specific binding of the agonist radioligand [¹²⁵I](±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane ([¹²⁵I]DOI) and the ability of 5′-guanylylimidodiphosphate to attenuate this binding, suggesting that at the early stage of antagonist-induced desensitization the capacity of the 5-HT_2A receptor to couple to G protein is impaired.