RESUMEN
Trimeric G protein signaling is a fundamental mechanism of cellular communication in eukaryotes. The core of this mechanism consists of activation of G proteins by the guanine-nucleotide exchange factor (GEF) activity of G protein coupled receptors. However, the duration and amplitude of G protein-mediated signaling are controlled by a complex network of accessory proteins that appeared and diversified during evolution. Among them, nonreceptor proteins with GEF activity are the least characterized. We recently found that proteins of the ccdc88 family possess a Gα-binding and activating (GBA) motif that confers GEF activity and regulates mammalian cell behavior. A sequence similarity-based search revealed that ccdc88 genes are highly conserved across metazoa but the GBA motif is absent in most invertebrates. This prompted us to investigate whether the GBA motif is present in other nonreceptor proteins in invertebrates. An unbiased bioinformatics search in Caenorhabditis elegans identified GBAS-1 (GBA and SPK domain containing-1) as a GBA motif-containing protein with homologs only in closely related worm species. We demonstrate that GBAS-1 has GEF activity for the nematode G protein GOA-1 and that the two proteins are coexpressed in many cells of living worms. Furthermore, we show that GBAS-1 can activate mammalian Gα-subunits and provide structural insights into the evolutionarily conserved determinants of the GBA-G protein interface. These results demonstrate that the GBA motif is a functional GEF module conserved among highly divergent proteins across evolution, indicating that the GBA-Gα binding mode is strongly constrained under selective pressure to mediate receptor-independent G protein activation in metazoans.
Asunto(s)
Evolución Biológica , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Multimerización de Proteína , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Evolución Molecular , Proteínas de Unión al GTP/química , Expresión Génica , Factores de Intercambio de Guanina Nucleótido , Modelos Moleculares , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Transducción de SeñalRESUMEN
D1-like and D2-like dopamine receptors have synergistic and antagonistic effects on behavior. To understand the mechanisms underlying these effects, we studied dopamine signaling genetically in Caenorhabditis elegans. Knocking out a D2-like receptor, DOP-3, caused locomotion defects similar to those observed in animals lacking dopamine. Knocking out a D1-like receptor, DOP-1, reversed the defects of the DOP-3 knockout. DOP-3 and DOP-1 have their antagonistic effects on locomotion by acting in the same motor neurons, which coexpress the receptors and which are not postsynaptic to dopaminergic neurons. In a screen for mutants unable to respond to dopamine, we identified four genes that encode components of the antagonistic Galpha(o) and Galpha(q) signaling pathways, including Galpha(o) itself and two subunits of the regulator of G protein signaling (RGS) complex that inhibits Galpha(q). Our results indicate that extrasynaptic dopamine regulates C. elegans locomotion through D1- and D2-like receptors that activate the antagonistic Galpha(q) and Galpha(o) signaling pathways, respectively.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Dopamina/metabolismo , Sistema Nervioso/metabolismo , Receptores de Dopamina D2/metabolismo , Receptores Dopaminérgicos/metabolismo , Transducción de Señal/fisiología , Acetilcolina/metabolismo , Animales , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/aislamiento & purificación , ADN Complementario/análisis , ADN Complementario/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/aislamiento & purificación , Proteínas de Unión al GTP/metabolismo , Marcación de Gen , Datos de Secuencia Molecular , Actividad Motora/genética , Neuronas Motoras/metabolismo , Mutación/genética , Filogenia , Proteínas RGS/genética , Proteínas RGS/metabolismo , Receptores Dopaminérgicos/genética , Receptores Dopaminérgicos/aislamiento & purificación , Receptores de Dopamina D1/genética , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/aislamiento & purificación , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Transducción de Señal/genéticaRESUMEN
A better understanding of the molecular mechanisms of signaling by the neurotransmitter serotonin is required to assess the hypothesis that defects in serotonin signaling underlie depression in humans. Caenorhabditis elegans uses serotonin as a neurotransmitter to regulate locomotion, providing a genetic system to analyze serotonin signaling. From large-scale genetic screens we identified 36 mutants of C. elegans in which serotonin fails to have its normal effect of slowing locomotion, and we molecularly identified eight genes affected by 19 of the mutations. Two of the genes encode the serotonin-gated ion channel MOD-1 and the G-protein-coupled serotonin receptor SER-4. mod-1 is expressed in the neurons and muscles that directly control locomotion, while ser-4 is expressed in an almost entirely non-overlapping set of sensory and interneurons. The cells expressing the two receptors are largely not direct postsynaptic targets of serotonergic neurons. We analyzed animals lacking or overexpressing the receptors in various combinations using several assays for serotonin response. We found that the two receptors act in parallel to affect locomotion. Our results show that serotonin functions as an extrasynaptic signal that independently activates multiple receptors at a distance from its release sites and identify at least six additional proteins that appear to act with serotonin receptors to mediate serotonin response.