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Título

Multiple conformations of native and recombinant human 5-hydroxytryptamine2A receptors are labeled by agonists and discriminated by antagonists

Autor López-Giménez, Juan F. ; Villazón, María; Brea, José Manuel; Loza, María Isabel; Palacios, José M.; Mengod Los Arcos, Guadalupe ; Vilaró, Maria Teresa
Fecha de publicación 2001
EditorAmerican Society for Pharmacology and Experimental Therapeutics
Citación Molecular Pharmacology 60(4): 690-699 (2001)
ResumenWe 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-HT2A receptor antagonists by using new selective antagonists of 5-HT2A (MDL100,907) and 5-HT2C (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-HT2A receptors (frontal cortex and striosomes). Furthermore, the effect of uncoupling receptor/G protein complexes on antagonist competition was studied with guanosine-5′-(β,γ-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-HT2A 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 (Kdn = 0.8nM, Kd1 = 31.22 nM). Addition of 100 μM Gpp(NH)p resulted in a single low-affinity (Kd = 24.44 nM) site with unchanged Bmax. [3H]5-HT showed no specific binding to 5-HT2A 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.
URI http://hdl.handle.net/10261/112688
Identificadoresissn: 0026-895X
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