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) re ceptor agonist (+/-)-1-(2,5-dimethoxy-4-[I-125]iodophenyl)-2-aminopropane [ (+/-)-[I-125]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 ketan serin and mesulergine. Autoradiographic competition experiments were perfor med with these antagonists in human brain regions where (+/-)-[I-125]DOI la bels almost exclusively 5-HT2A receptors (frontal cortex and striosomes). F urthermore, the effect of uncoupling receptor/G protein complexes on antago nist competition was studied with guanosine-5'-(beta,gamma -imido)triphosph ate [Gpp(NH)p]. Competition experiments with (+/-)-[H-3]1-(4-bromo-2,5-dime thoxyphenil)-2-aminopropane [(+/-)-[H-3]DOB] were also performed in membran es from Chinese hamster ovary cells (CHOFA4) expressing cloned human 5-HT2A receptors. In both systems, ketanserin and MDL100,907 displayed biphasic c ompetition profiles, whereas SB242084 and mesulergine competed monophasical ly. In absence of antagonist, 100 muM Gpp(NH)p decreased brain (+/-)-[I-125 ]DOI specific binding by 40 to 50% and (+/-)-[H-3]DOB specific binding to C HOFA4 cells by 30%. The remaining agonist-labeled uncoupled sites were stil l displaced biphasically by ketanserin and MDL100,907, with unaltered affin ities. Saturation experiments were performed in CHOFA4 cells. (+/-)-[H-3]DO B labeled two sites (K-dh = 0.8 nM, K-dl = 31.22 nM). Addition of 100 muM G pp(NH)p resulted in a single low-affinity (K-d = 24.44 nM) site with unchan ged B-max. [H-3]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 agoni sts possibly label all three conformations and ketanserin and MDL100,907 re cognize with different affinities at least two of these conformations.