Multiple conformations of native and recombinant human 5-hydroxytryptamine(2a) receptors are labeled by agonists and discriminated by antagonists.
We have expanded previous studies with the 5-hydroxytryptamine (5-HT)(2) receptor agonist (+/-)-1-(2,5-dimethoxy-4-[(125)I]iodophenyl)-2-aminopropane [(+/-)-[(125)I]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 (+/-)-[(125)I]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'-(beta,gamma-imido)triphosphate [Gpp(NH)p]. Competition experiments with (+/-)-[(3)H]1-(4-bromo-2,5-dimethoxyphenil)-2-aminopropane [(+/-)-[(3)H]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 microM Gpp(NH)p decreased brain (+/-)-[(125)I]DOI specific binding by 40 to 50% and (+/-)-[(3)H]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. (+/-)-[(3)H]DOB labeled two sites (K(d(h))= 0.8 nM, K(d(l)) = 31.22 nM). Addition of 100 microM Gpp(NH)p resulted in a single low-affinity (K(d) = 24.44 nM) site with unchanged B(max). [(3)H]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.
López-Giménez
J F
JF
Department of Neurochemistry, Instituto de Investigaciones Biomédicas de Barcelona-Consejo Superior de Investigaciones Cientificas, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Barcelona, Spain.
Villazón
M
M
Brea
J
J
Loza
M I
MI
Palacios
J M
JM
Mengod
G
G
Vilaró
M T
MT
D000818:Animals; D001345:Autoradiography; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D005464:Fluorobenzenes / Q000494:pharmacology; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D010880:Piperidines / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D044402:Receptor, Serotonin, 5-HT2A; D011985:Receptors, Serotonin / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D012702:Serotonin Antagonists / Q000494:pharmacology; D017366:Serotonin Receptor Agonists / Q000494:pharmacology; D014162:Transfection; D014316:Tritium
D005464:Fluorobenzenes; D007295:Inositol Phosphates; D010880:Piperidines; D044402:Receptor, Serotonin, 5-HT2A; D011985:Receptors, Serotonin; D011994:Recombinant Proteins; D012702:Serotonin Antagonists; D017366:Serotonin Receptor Agonists; D014316:Tritium; C079049:volinanserin