Vol. 60, Issue 4, 690-699, October 2001

Multiple Conformations of Native and Recombinant Human 5-Hydroxytryptamine_2A Receptors Are Labeled by Agonists and Discriminated by Antagonists

Juan F. López-Giménez, María Villazón, José Brea, M. Isabel Loza, José M. Palacios,¹ Guadalupe Mengod, and M. Teresa Vilaró

Department of Neurochemistry, Instituto de Investigaciones Biomédicas de Barcelona-Consejo Superior de Investigaciones Cientificas (Institut d'Investigacions Biomèdiques August Pi i Sunyer), Barcelona, Spain (J.F.L.-G., G.M., M.T.V.); and Department of Pharmacology, Universidad de Santiago de Compostela, Santiago de Compostela, Spain (M.V., J.B., M.I.L.)

We have expanded previous studies with the 5-hydroxytryptamine (5-HT)₂ receptor agonist (±)-1-(2,5-dimethoxy-4-[¹²⁵I]iodophenyl)-2-aminopropane [(±)-[¹²⁵I]DOI] in human brain that had shown biphasic competition curves for several 5-HT_2A receptor antagonists by using new selective antagonists of 5-HT_2A (MDL100,907) and 5-HT_2C (SB242084) receptors together with ketanserin and mesulergine. Autoradiographic competition experiments were performed with these antagonists in human brain regions where (±)-[¹²⁵I]DOI labels almost exclusively 5-HT_2A receptors (frontal cortex and striosomes). Furthermore, the effect of uncoupling receptor/G protein complexes on antagonist competition was studied with guanosine-5'-(,-imido)triphosphate [Gpp(NH)p]. Competition experiments with (±)-[³H]1-(4-bromo-2,5-dimethoxyphenil)-2-aminopropane [(±)-[³H]DOB] were also performed in membranes from Chinese hamster ovary cells (CHOFA4) expressing cloned human 5-HT_2A receptors. In both systems, ketanserin and MDL100,907 displayed biphasic competition profiles, whereas SB242084 and mesulergine competed monophasically. In absence of antagonist, 100 µM Gpp(NH)p decreased brain (±)-[¹²⁵I]DOI specific binding by 40 to 50% and (±)-[³H]DOB specific binding to CHOFA4 cells by 30%. The remaining agonist-labeled uncoupled sites were still displaced biphasically by ketanserin and MDL100,907, with unaltered affinities. Saturation experiments were performed in CHOFA4 cells. (±)-[³H]DOB labeled two sites (K_dh= 0.8 nM, K_dl = 31.22 nM). Addition of 100 µM Gpp(NH)p resulted in a single low-affinity (K_d = 24.44 nM) site with unchanged B_max. [³H]5-HT showed no specific binding to 5-HT_2A receptors. These results conform with the extended ternary complex model of receptor action that postulates the existence of partly activated receptor conformation(s) (R*) in equilibrium with the ground (R) and the activated G protein-coupled (R*G) conformations. Thus, both in human brain and CHOFA4 cells, the agonists possibly label all three conformations and ketanserin and MDL100,907 recognize with different affinities at least two of these conformations.