RESUMO
BACKGROUND: Gbeta proteins have traditionally been thought to complex with Ggamma proteins to function as subunits of G protein heterotrimers. The divergent Gbeta(5) protein, however, can bind either Ggamma proteins or regulator of G protein signaling (RGS) proteins that contain a G gamma-like (GGL) domain. RGS proteins inhibit G protein signaling by acting as Galpha GTPase activators. While Gbeta(5) appears to bind RGS proteins in vivo, its association with Ggamma proteins in vivo has not been clearly demonstrated. It is unclear how Gbeta(5) might influence RGS activity. In C. elegans there are exactly two GGL-containing RGS proteins, EGL-10 and EAT-16, and they inhibit Galpha(o) and Galpha(q) signaling, respectively. RESULTS: We knocked out the gene encoding the C. elegans Gbeta(5) ortholog, GPB-2, to determine its physiological roles in G protein signaling. The gpb-2 mutation reduces the functions of EGL-10 and EAT-16 to levels comparable to those found in egl-10 and eat-16 null mutants. gpb-2 knockout animals are viable, and exhibit no obvious defects beyond those that can be attributed to a reduction of EGL-10 or EAT-16 function. GPB-2 protein is nearly absent in eat-16; egl-10 double mutants, and EGL-10 protein is severely diminished in gpb-2 mutants. CONCLUSIONS: Gbeta(5) functions in vivo complexed with GGL-containing RGS proteins. In the absence of Gbeta(5), these RGS proteins have little or no function. The formation of RGS-Gbeta(5) complexes is required for the expression or stability of both the RGS and Gbeta(5) proteins. Appropriate RGS-Gbeta(5) complexes regulate both Galpha(o) and Galpha(q) proteins in vivo.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/fisiologia , Reguladores de Proteínas de Ligação ao GTP , Subunidades beta da Proteína de Ligação ao GTP , Proteínas de Ligação ao GTP/fisiologia , Proteínas de Helminto/metabolismo , Proteínas de Helminto/fisiologia , Proteínas Heterotriméricas de Ligação ao GTP/antagonistas & inibidores , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Proteínas RGS/fisiologia , Transdução de Sinais/fisiologia , Animais , Animais Geneticamente Modificados , Comportamento Animal , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Expressão Gênica , Proteínas de Helminto/genética , Proteínas Heterotriméricas de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/fisiologia , Camundongos , Neurônios/metabolismo , Oviposição , Proteínas RGS/genética , Proteínas RGS/metabolismo , TransgenesRESUMO
Regulators of G protein signaling (RGS proteins) inhibit heterotrimeric G protein signaling by activating G protein GTPase activity. Many mammalian RGS proteins are expressed in the brain and can act in vitro on the neural G protein G(o), but the biological purpose of this multiplicity of regulators is not clear. We have analyzed all 13 RGS genes in Caenorhabditis elegans and found that three of them influence the aspect of egg-laying behavior controlled by G(o) signaling. A previously studied RGS protein, EGL-10, affects egg laying under all conditions tested. The other two RGS proteins, RGS-1 and RGS-2, act as G(o) GTPase activators in vitro but, unlike EGL-10, they do not strongly affect egg laying when worms are allowed to feed constantly. However, rgs-1; rgs-2 double mutants fail to rapidly induce egg-laying behavior when refed after starvation. Thus EGL-10 sets baseline levels of signaling, while RGS-1 and RGS-2 appear to redundantly alter signaling to cause appropriate behavioral responses to food.
Assuntos
Comportamento Animal , Caenorhabditis elegans/fisiologia , Alimentos , Proteínas de Ligação ao GTP/metabolismo , Proteínas RGS/fisiologia , Transdução de Sinais , Sequência de Aminoácidos , Animais , Proteínas de Ligação ao GTP/genética , Dados de Sequência Molecular , Mutação , Sistema Nervoso/metabolismo , Fenótipo , Proteínas RGS/genética , Homologia de Sequência de AminoácidosRESUMO
Cocrystal structures of wild-type TATA box-binding protein (TBP) recognizing 10 naturally occurring TATA elements have been determined at 2.3-1.8 A resolution, and compared with our 1.9 A resolution structure of TBP bound to the Adenovirus major late promoter (AdMLP) TATA box (5'-TATAAAAG-3'). Minor-groove recognition by the saddle-shaped protein induces the same conformational change in each of these oligonucleotides, despite variations in promoter sequence that reduce the efficiency of transcription initiation. Three molecular mechanisms explain assembly of diverse TBP-TATA element complexes. (1) T --> A and A --> T transversions leave the minor-groove face unchanged, permitting formation of TBP-DNA complexes on many A/T-rich core promoter sequences. (2) Cavities in the interface between TBP and the minor-groove face of the AdMLP TATA box accommodate the exocyclic NH(2) groups of G in a TACA box and in a TATAAG box. (3) Formation of a C:G Hoogsteen basepair in a TATAAAC box eliminates steric clashes that would be produced by the Watson-Crick base pair. We conclude that the structure of the TBP-TATA box complex found at the heart of the polymerase II (pol II) transcription machinery has remained constant over the course of evolution, despite variations in TBP and its DNA targets.
