[
BMC Genomics,
2007]
ABSTRACT: BACKGROUND: In the genome of Caenorhabditis elegans, homopolymeric poly-G/poly-C tracts (G/C tracts) exist at high frequency and are maintained by the activity of the DOG-1 protein. The frequency and distribution of G/C tracts in the genomes of C. elegans and the related nematode, C. briggsae were analyzed to investigate possible biological roles for G/C tracts. RESULTS: In C. elegans, G/C tracts are distributed along every chromosome in a non-random pattern. Most G/C tracts are within introns or are close to genes. Analysis of SAGE data showed that G/C tracts correlate with the levels of regional gene expression in C. elegans. G/C tracts are over-represented and dispersed across all chromosomes in another Caenorhabditis species, C. briggase. However, the positions and distribution of G/C tracts in C. briggsae differ from those in C. elegans. Furthermore, the C. briggsae
dog-1 ortholog CBG19723 can rescue the mutator phenotype of C. elegans
dog-1 mutants. CONCLUSIONS: The abundance and genomic distribution of G/C tracts in C. elegans, the effect of G/C tracts on regional transcription levels, and the lack of positional conservation of G/C tracts in C. briggsae suggest a role for G/C tracts in chromatin structure but not in the transcriptional regulation of specific genes.
[
Genes Dev,
1999]
To elucidate the cellular role of the heterotrimeric G protein G(o), we have taken a molecular genetic approach in Caenorhabditis elegans. We screened for suppressors of activated GOA-1 (G(o)alpha) that do not simply decrease its expression and found mutations in only two genes,
sag-1 and
eat-16. Animals defective in either gene display a hyperactive phenotype similar to that of
goa-1 loss-of-function mutants. Double-mutant analysis indicates that both
sag-1 and
eat-16 act downstream of, or parallel to, G(o)alpha and negatively regulate EGL-30 (G(q)alpha) signaling.
eat-16 encodes a regulator of G protein signaling (RGS) most similar to the mammalian RGS7 and RGS9 proteins and can inhibit endogenous mammalian G(q)/G(11) in COS-7 cells. Animals defective in both
sag-1 and
eat-16 are inviable, but reducing function in
egl-30 restores viability, indicating that the lethality of the
eat-16;
sag-1 double mutant is due to excessive G(q)alpha activity. Analysis of these mutations indicates that the G(o) and G(q) pathways function antagonistically in C. elegans, and that G(o)alpha negatively regulates the G(q) pathway, possibly via EAT-16 or SAG-1. We propose that a major cellular role of G(o) is to antagonize signaling by G(q).
[
WormBook,
2006]
Heterotrimeric G proteins, composed of alpha , beta , and gamma subunits, are able to transduce signals from membrane receptors to a wide variety of intracellular effectors. In this role, G proteins effectively function as dimers since the signal is communicated either by the G alpha subunit or the stable G betagamma complex. When inactive, G alpha -GDP associates with G betagamma and the cytoplasmic portion of the receptor. Ligand activation of the receptor stimulates an exchange of GTP for GDP resulting in the active signaling molecules G alpha -GTP and free G betagamma , either of which can interact with effectors. Hydrolysis of GTP restores G alpha -GDP, which then reassociates with G betagamma and receptor to terminate signaling. The rate of G protein activation can be enhanced by the guanine-nucleotide exchange factor, RIC-8 , while the rate of GTP hydrolysis can be enhanced by RGS proteins such as EGL-10 and EAT-16 . Evidence for a receptor-independent G-protein-signaling pathway has been demonstrated in C. elegans early embryogenesis. In this pathway, the G alpha subunits GOA-1 and GPA-16 are apparently activated by the non-transmembrane proteins GPR-1 , GPR-2 , and RIC-8 , and negatively regulated by RGS-7 . The C. elegans genome encodes 21 G alpha , 2 G beta and 2 G gamma subunits. The alpha subunits include one ortholog of each mammalian G alpha family: GSA-1 (Gs), GOA-1 (Gi/o), EGL-30 (Gq) and GPA-12 (G12). The remaining C. elegans alpha subunits ( GPA-1 , GPA-2 , GPA-3 , GPA-4 , GPA-5 , GPA-6 , GPA-7 , GPA-8 , GPA-9 , GPA-10 , GPA-11 , GPA-13 , GPA-14 , GPA-15 , GPA-16 , GPA-17 and ODR-3 ) are most similar to the Gi/o family, but do not share sufficient homology to allow classification. The conserved G alpha subunits, with the exception of GPA-12 , are expressed broadly while 14 of the new G alpha genes are expressed in subsets of chemosensory neurons. Consistent with their expression patterns, the conserved C. elegans alpha subunits, GSA-1 , GOA-1 and EGL-30 are involved in diverse and fundamental aspects of development and behavior. GOA-1 acts redundantly with GPA-16 in positioning of the mitotic spindle in early embryos. EGL-30 and GSA-1 are required for viability starting from the first larval stage. In addition to their roles in development and behaviors such as egg laying and locomotion, the EGL-30 , GSA-1 and GOA-1 pathways interact in a network to regulate acetylcholine release by the ventral cord motor neurons. EGL-30 provides the core signals for vesicle release, GOA-1 negatively regulates the EGL-30 pathway, and GSA-1 modulates this pathway, perhaps by providing positional cues. Constitutively activated GPA-12 affects pharyngeal pumping. The G alpha subunits unique to C. elegans are primarily involved in chemosensation. The G beta subunit, GPB-1 , as well as the G gamma subunit, GPC-2 , appear to function along with the alpha subunits in the classic G protein heterotrimer. The remaining G beta subunit, GPB-2 , is thought to regulate the function of certain RGS proteins, while the remaining G gamma subunit, GPC-1 , has a restricted role in chemosensation. The functional difference for most G protein pathways in C. elegans, therefore, resides in the alpha subunit. Many cells in C. elegans express multiple G alpha subunits, and multiple G protein pathways are known to function in specific cell types. For example, Go, Gq and Gs-mediated signaling occurs in the ventral cord motor neurons. Similarly, certain amphid neurons use multiple G protein pathways to both positively and negatively regulate chemosensation. C. elegans thus provides a powerful model for the study of interactions between and regulation of G protein signaling.
[
East Coast Worm Meeting,
2004]
Defects in signaling by the neurotransmitter dopamine underlie schizophrenia and drug addiction and loss of dopamine signaling causes Parkinson's disease. Dopamine controls the activity of neurons by acting through two classes of G protein-coupled receptors known as D1-like and D2-like. Activation of D1- and D2-like receptors have opposing effects on behavior, but the signaling mechanisms underlying this antagonism remain debated. We analyzed the mechanisms of dopamine signaling genetically in C. elegans . Knocking out a D2-like receptor, DOP-3, caused behavioral defects similar to those observed in animals lacking dopamine. Knocking out a D1-like receptor, DOP-1, reversed the defects seen in the DOP-3 knockout. Thus in C. elegans , as in mammals, D1- and D2-like signaling antagonize each other to control behavior. We identified the physiological signaling pathways responsible for this antagonism in C. elegans using a genetic screen for mutants unable to respond to dopamine. The screen identified four genes that encode components of the antagonistic G a o and G a q signaling pathways, including G a o itself and two subunits of the RGS complex that inhibits G a q . Thus dopamine regulates behavior in C. elegans through D1- and D2-like receptors that activate the antagonistic G a q and G a o signaling pathways, respectively. While the antagonistic G a q and G a o signaling pathways have been well-characterized in C. elegans this mechanism has not been previously implicated in dopamine signaling. Each of the signaling components identified in C. elegans is conserved in mammals and expressed in the brain. If the mechanism of G a q and G a o signaling is also conserved from C. elegans to mammals, signaling by D1-like receptors through G a q and D2-like receptors through G a o could explain many of the observed antagonistic effects of these receptors in mammals.
[
Curr Biol,
2001]
In C. elegans, a G(o)/G(q) signaling network regulates locomotion and egg laying [1-8]. Genetic analysis shows that activated Ca2+/calmodulin-dependent protein kinase II (CaMKII) is suppressed by perturbations of this network, which include loss of the GOA-1 G(o)alpha, DGK-1 diacylglycerol kinase. EAT-16 16 protein gamma subunit-like (GGL)-containing RGS protein, or an unidentified protein encoded by the gene
eat-11 [9]. We cloned
eat-11 and report that it encodes the G beta (5) ortholog GPB-2, Gp, binds specifically to GGL-containing RGS proteins, and the G beta (5)/RGS complex can promote the GTP-hydrolyzing activity of G alpha subunits [10, 11]. However, little is known about how this interaction affects G protein signaling in vivo. In addition to EAT-16, the GGL-containing RGS protein EGL-10 participates in G(o)/G(q) signaling; EGL-10 appears to act as an RGS for the GOA-1 G,cw, while EAT-16 appears to act as an RGS for the EGL-30 G(q)alpha [4, 5]. We have combined behavioral, electrophysiological, and pharmacological approaches to show that GPB-2 is a central member of the G(o)/G(q) network and that GPB-2 may interact with both the EGL-10 and EAT-16 RGS proteins to mediate the opposing activities of G,cw and G,a. These interactions provide a mechanism for the modulation of behavior by antagonistic G protein networks.
[
International C. elegans Meeting,
2001]
Gbeta proteins have traditionally been thought to complex with G g proteins to function as subunits of G protein heterotrimers. The divergent Gbeta 5 protein, however, can bind either G g proteins or r egulator of G protein s ignaling (RGS) proteins that contain a G - g amma- l ike (GGL) domain. RGS proteins are inhibitors of G protein signaling that act as G a GTPase activators. While Gbeta 5 appears to bind RGS proteins in vivo , its association with G g 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 G a o and G a q signaling, respectively. 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. These results indicate that 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 G a o and G a q proteins in vivo .
[
Genes Dev,
1999]
A wide variety of extracellular stimuli induce signal transduction through receptors coupled to heterotrimeric G proteins, which consist of alpha, beta, and gamma subunits (Gilman 1987). The G alpha subunit has guanine nucleotide binding and GTP hydrolysis activities. Based on function and amino acid sequence homology, the Galpha, G alph i/o, G alpha q, and G alpha 12 (Simon et al. 1991; Hepler and Gilman 1992). As exemplified by the responsiveness of our five senses to environmental stimuli, signaling mediated by trimeric G proteins is often extremely rapid and transient. A key step in achieving such a raid response is the ability of the G alpha subunit to switch between it GDP- and GTP-bound forms. The nucleotide binding state of G alpha is regulated at both the GDP dissociation and GTP hydrolysis steps. Stimulation of receptors by agonists leads to a conformational change in the receptors which can function as a guanine nucleotide exchange factor to stimulate a rapid dissociation of GDP from the inactive G alpha. The nucleotide-free G alpha is then available to bind GTP, leading to the dissociation of G alpha from the G beta gamma heterodimer. Both the G alpha and G beat gamma subunits can interact with and regulate downstream effectors that include adenylyl cyclase, phospholipase C, and ion channels (Gilman 1987; Birnbaumer 1992).
[
Curr Biol,
2001]
Background: Gp proteins have traditionally been thought to complex with Gy proteins to function as subunits of G protein heterotrimers. The divergent G beta (5) protein, however, can bind either Gy 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 Ga GTPase activators. While GP, appears to bind RGS proteins in vivo, its association with Gy proteins in vivo has not been clearly demonstrated. It is unclear how Gp, might influence RGS activity, In C. elegans there are exactly two GGL-containing RGS proteins, EGL-10 and EAT-16, and they inhibit G alpha (o) and G alpha (q) signaling, respectively. Results: We knocked out the gene encoding the C. elegans G beta (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: G beta (5) functions in vivo complexed with GGL-containing RGS proteins. In the absence of G beta (5), these RGS proteins have little or no function. The formation of RGS-G beta (5) complexes is required for the expression or stability of both the RGS and G beta (5) proteins. Appropriate RGS-G beta (5) complexes regulate both G alpha (o) and G alpha (q) proteins in vivo.