RESUMEN
We report how rotational variations in transmembrane (TM) helix interactions participate in the activity states of the thrombopoietin receptor (TpoR), a type 1 cytokine receptor that controls the production of blood platelets. We also explore the mechanism of small-molecule agonists that do not mimic the natural ligand. We show, by a combination of cysteine cross-linking, alanine-scanning mutagenesis, and computational simulations, that the TpoR TM dimerizes strongly and can adopt 3 different stable, rotationally related conformations, which may correspond to specific states of the full-length receptor (active, inactive, and partially active). Thus, our data suggest that signaling and inactive states of the receptor are related by receptor subunit rotations, rather than a simple monomer-dimer transition. Moreover, results from experiments with and without agonists in vitro and in cells allow us to propose a novel allosteric mechanism of action for a class of small molecules, in which they activate TpoR by binding to the TM region and by exploiting the rotational states of the dimeric receptor. Overall, our results support the emerging view of the participation of mutual rotations of the TM domains in cytokine receptor activation.
Asunto(s)
Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores de Trombopoyetina/química , Regulación Alostérica , Secuencia de Aminoácidos , Simulación por Computador , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Mutación , Piperidinas/química , Piperidinas/farmacología , Conformación Proteica , Receptores de Trombopoyetina/agonistas , Receptores de Trombopoyetina/genética , RotaciónRESUMEN
Pathogenesis of cholera diarrhea requires cholera toxin (CT)-mediated adenosine diphosphate (ADP)-ribosylation of stimulatory G protein (Gsα) in enterocytes. CT is an AB5 toxin with an inactive CTA1 domain linked via CTA2 to a pentameric receptor-binding B subunit. Allosterically activated CTA1 fragment in complex with NAD+ and GTP-bound ADP-ribosylation factor 6 (ARF6-GTP) differs conformationally from the CTA1 domain in holotoxin. A surface-exposed knob and a short α-helix (formed, respectively, by rearranging "active-site" and "activation" loops in inactive CTA1) and an ADP ribosylating turn-turn (ARTT) motif, all located near the CTA1 catalytic site, were evaluated for possible roles in recognizing Gsα. CT variants with one, two or three alanine substitutions at surface-exposed residues within these CTA1 motifs were tested for assembly into holotoxin and ADP-ribosylating activity against Gsα and diethylamino-(benzylidineamino)-guanidine (DEABAG), a small substrate predicted to fit into the CTA1 active site). Variants with single alanine substitutions at H55, R67, L71, S78, or D109 had nearly wild-type activity with DEABAG but significantly decreased activity with Gsα, suggesting that the corresponding residues in native CTA1 participate in recognizing Gsα. As several variants with multiple substitutions at these positions retained partial activity against Gsα, other residues in CTA1 likely also participate in recognizing Gsα.
Asunto(s)
Toxina del Cólera/genética , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/genética , Factores de Ribosilacion-ADP/metabolismo , Animales , Dominio Catalítico , Línea Celular , Toxina del Cólera/metabolismo , Análisis Mutacional de ADN , Variación Genética , Ratones , Modelos Moleculares , Unión ProteicaRESUMEN
The ligand-gated ion channel family includes receptors for serotonin (5-hydroxytryptamine, 5-HT), acetylcholine, GABA, and glutamate. Drugs targeting subtypes of these receptors have proven useful for the treatment of various neuropsychiatric and neurological disorders. To identify new ligand-gated ion channels as potential therapeutic targets, drafts of human genome sequence were interrogated. Portions of four novel genes homologous to 5-HT(3A) and 5-HT(3B) receptors were identified within human sequence databases. We named the genes 5-HT(3C1)-5-HT(3C4). Radiation hybrid (RH) mapping localized these genes to chromosome 3q27-28. All four genes shared similar intron-exon organizations and predicted protein secondary structure with 5-HT(3A) and 5-HT(3B). Orthologous genes were detected by Southern blotting in several species including dog, cow, and chicken, but not in rodents, suggesting that these novel genes are not present in rodents or are very poorly conserved. Two of the novel genes are predicted to be pseudogenes, but two other genes are transcribed and spliced to form appropriate open reading frames. The 5-HT(3C1) transcript is expressed almost exclusively in small intestine and colon, suggesting a possible role in the serotonin-responsiveness of the gut.