G protein salpha 60A



Following gastrulation, G salpha reactivity increases; by germband extension [Images], it is associated with all cell membranes. After completion of germband retraction, elevated levels of G salpha and G oalpha are first detected in the forming neuropil of the brain and ventral ganglion. This pattern persists for the duration of embryogenesis and through adult life (Wolfgang, 1991).


Messenger RNA of Drosophila G salpha is located in the cortex of the brain, which contains the neuronal cell bodies. Little or none is detected in the central neuropil, which contains neurites and synapses but no cell bodies. An intermediate level is present in the eyes. The cortex of the optic lobes have consistently lower levels than the midbrain cortex. In addition, mRNA is present in the cortex of the ventral ganglion (Quan, 1989). No Gs alpha immunoreactivity is observed in oocytes or nurse cells but is easily detected in the cortex of follicle cells and presumably is associated with cell membranes (Wolfgang, 1991).

G oalpha and G salpha homologs are expressed at highest levels in neuropils and at intermediate levels in the cortex of all brain and thoracic ganglion areas. Only the lamina in the CNS contains low levels of these alpha-subunits. Additionally, G salpha appears to be associated with the cell membranes of neuronal cell bodies, while G oalpha has a more diffuse distribution, suggesting its presence in the cytoplasm as well as cell membranes. In contrast to the wide distribution of G oalpha and G salpha, G ialpha has a surprisingly restricted distribution in the CNS. It is present at high levels only in photoreceptor cell terminations, the glomerulae of the antennal lobes, and the ocellar retina. Little or no G ialpha is detected in other brain regions or in the thoracic ganglion. G ialpha, then, appears to be uniquely associated with some primary sensory afferents and their terminations, suggesting the presence of specific receptor and/or effector systems which mediate the transmission of primary sensory information in Drosophila (Wolfgang, 1990).

Drosophila pacemaker neurons require g protein signaling and GABAergic inputs to generate twenty-four hour behavioral rhythms

Intercellular signaling is important for accurate circadian rhythms. In Drosophila, the small ventral lateral neurons (s-LNvs) are the dominant pacemaker neurons and set the pace of most other clock neurons in constant darkness. This study shows that two distinct G protein signaling pathways are required in LNvs for 24 hr rhythms. Reducing signaling in LNvs via the G alpha subunit Gs, which signals via cAMP, or via the G alpha subunit Go, which signals via Phospholipase 21c, lengthens the period of behavioral rhythms. In contrast, constitutive Gs or Go signaling makes most flies arrhythmic. Using dissociated LNvs in culture, it was found that Go and the metabotropic GABA(B)-R3 receptor are required for the inhibitory effects of GABA on LNvs and that reduced GABA(B)-R3 expression in vivo lengthens period. Although no clock neurons produce GABA, hyperexciting GABAergic neurons disrupts behavioral rhythms and s-LNv molecular clocks. Therefore, s-LNvs require GABAergic inputs for 24 hr rhythms (Dahdal, 2010).

The long-periods observed with reduced Gs signaling are consistent with four other manipulations of cAMP levels or PKA activity that alter fly circadian behavior. First, long-period rhythms with dnc over-expression complement the short periods of dnc hypomorphs and suggest that the latter are due to loss of dnc from LNvs. dnc mutants also increase phase shifts to light in the early evening. However, this study found no difference in phase delays or advances between Pdf > dnc and control flies, suggesting that altered light-responses of dnc hypomorphs are due to dnc acting in other clock neurons. The period-altering effects seen when manipulating cAMP levels are also consistent with finding stat expressing the cAMP-binding domain of mammalian Epac1 in LNvs lengthens period. This Epac1 domain likely reduces free cAMP levels in LNvs, although presumably not as potently as UAS-dnc. Third, mutations in PKA catalytic or regulatory subunits that affect the whole fly disrupt circadian behavior. Fourth, over-expressing a PKA catalytic subunit in LNvs rescues the period-altering effect of a UAS-shibire transgene that alters vesicle recycling, although the PKA catalytic subunit had no effect by itself. The long periods observed with reduced Gs signaling in LNvs also parallel mammalian studies in which pharmacologically reducing Adenylate cyclase activity lengthened period in SCN explants and mice (Dahdal, 2010).

G-proteins typically transduce extracellular signals. What signals could activate Gs in s-LNvs? PDF is one possibility since PDFR induces cAMP signaling in response to PDF in vitro, indicating that it likely couples to Gs. PDF could signal in an autocrine manner since PDFR is present in LNvs. However, the long-periods observed with reduced Gs signaling differ from the short-period and arrhythmic phenotypes of Pdf and pdfr mutants. The likeliest explanation for these differences is that the altered behavior of Pdf and pdfr mutants results from effects of PDF signaling over the entire circadian circuit, whereas the current manipulations specifically targeted LNvs. Indeed, LNvs are not responsible for the short-period rhythms in Pdf01 null mutant flies. Other possible explanations for the differences between the long-period rhythms with decreased Gs signaling in LNvs and the short-period rhythms of Pdf and pdfr mutants are that additional GPCRs couple to Gs in s-LNvs and influence molecular clock speed and that the current manipulations decrease rather than abolish reception of PDF. In summary, the data shows that Gs signaling via cAMP in s-LNvs modulates period length (Dahdal, 2010).

Go signaling via PLC21C constitutes a novel pathway that regulates the s-LNv molecular clock. This study found that Go and the metabotropic GABAB-R3 receptor are required for the inhibitory effects of GABA on larval LNvs, which develop into adult s-LNvs. The same genetic manipulations that block GABA inhibition of LNvs in culture (expression of Ptx or GABAB-R3-RNAi) lengthened the period of adult locomotor rhythms. Furthermore, the molecular clock in s-LNvs is disrupted when a subset of GABAergic neurons are hyper-excited. Since the LNvs do not produce GABA themselves, s-LNvs require GABAergic inputs to generate 24hr rhythms. Thus s-LNvs are less autonomous for determining period length in DD than previously anticipated (Dahdal, 2010).

Activation of G-proteins can have both short- and long-term effects on a cell. With Go signaling blocked by Ptx, short-term effects on LNv responses were detected in response to excitatory ACh and longer-term effects on the molecular clock. The latter are presumably explained by PLC activation since the behavioral phenotypes of Pdf > GoGTP flies were rescued by reducing Plc21C expression (Dahdal, 2010).

Since s-LNv clocks were unchanged even when the speed of all non-LNv clock neurons were genetically manipulated, it is surprising to find s-LNv clocks altered by signaling from GABAergic non-clock neurons. Why would LNvs need inputs from non-clock neurons to generate 24hr rhythms? One possibility is that LNvs receive multiple inputs which either accelerate or slow down the pace of their molecular clock but overall balance each other to achieve 24hr rhythms in DD. Since reducing signaling by Gs and Go lengthens period, these pathways normally accelerate the molecular clock. According to this model, there are unidentified inputs to LNvs which delay the clock. Identifying additional receptors in LNvs would allow this idea to be tested (Dahdal, 2010).

Previous work showed that GABAergic neurons project to LNvs and that GABAA receptors in l-LNvs regulate sleep. The current data show that constitutive activation of Go signaling dramatically alters behavioral rhythms, suggesting that LNvs normally receive rhythmic GABAergic inputs. But how can s-LNvs integrate temporal information from non clock-containing GABAergic neurons? s-LNvs could respond rhythmically to a constant GABAergic tone by controlling GABAB-R3 activity. Indeed, a recent study found that GABAB-R3 RNA levels in s-LNvs are much higher at ZT12 than at ZT0 (Kula-Eversole, 2010). Strikingly, this rhythm in GABAB-R3 expression is in antiphase to LNv neuronal activity. Thus regulated perception of inhibitory GABAergic inputs could at least partly underlie rhythmic LNv excitability. GABAergic inputs could also help synchronize LNvs as in the cockroach circadian system. Thus GABA's short-term effects on LNv excitability, likely mediated by Gβ/γ, and GABA's longer-term effects on the molecular clock via Go may both contribute to robust rhythms (Dahdal, 2010).

This work adds to the growing network view of circadian rhythms in Drosophila where LNvs integrate information to set period for the rest of the clock network in DD. The period-altering effects of decreased G-protein signaling in LNvs point to a less hierarchical and more distributed network than previously envisioned. Since the data strongly suggests that GABA inputs are novel regulators of 24hr rhythms, the GABAergic neurons that fine-tune the s-LNv clock should be considered part of the circadian network (Dahdal, 2010).

Effects of Mutation or Deletion

One of the best understood signal transduction pathways activated by receptors containing seven transmembrane domains involves activation of heterotrimeric G-protein complexes containing Gsalpha, the subsequent stimulation of adenylyl cyclase, production of cAMP, activation of protein kinase A (PKA), and the phosphorylation of substrates that control a wide variety of cellular responses. The identification of 'loss-of-function' mutations in the Drosophila Gsalpha gene (dgs) is reported in this study. Seven mutants have been identified that are either complemented by transgenes representing the wild-type dgs gene or contain nucleotide sequence changes resulting in the production of altered Gsalpha protein. Examination of mutant alleles representing loss-of-Gsalpha function indicates that the phenotypes generated do not mimic those created by mutational elimination of PKA. These results are consistent with the conclusion reached in previous studies that activation of PKA, at least in these developmental contexts, does not depend on receptor-mediated increases in intracellular cAMP, in contrast to the predictions of models developed primarily on the basis of studies in cultured cells (Wolfgang, 2001).

Since loss-of-function mutations in genes encoding Gsalpha in both mice and C. elegans result in lethality, an F2 lethal screen was performed to recover mutations in the gene (dgs) encoding the Drosophila Gsalpha protein. The screen was based on the observation that immunoblots of extracts prepared from Df(2R)orBR11, cn, bw, sp/SM6, eve-lacz, Roi flies probed with Gsalpha-specific antibodies had approximately half the amount of immunoreactive protein compared to controls, suggesting that this deficiency eliminates the dgs gene. Thus, male cn;ry506, es flies were mutagenized with EMS and crossed en masse to CyO/Gla females. Single F1 CyO or Gla males were then mated to virgin Df(2R)orBR11, cn, bw, sp/SM6, eve-lacz, Roi females. From roughly 4000 such crosses, 120 balanced mutations were recovered that were lethal over this deficiency. Of these, 47 were excluded from further consideration since they were lethal over In(2LR) ltG10/Cy Roi, an overlapping deficiency that does not remove dgs as assessed by immunoblot analysis. The 73 remaining balanced lethal mutations were crossed to deficiency stocks containing a dgs rescue transgene Gs27; df(2R) orBR11, cn, bw, sp/CyO. Seven of the lethals were shown to be in the same complementation group and all but dgsR19 (which carries a linked, second site recessive lethal mutation) were rescued by the Gs27 transgene. These are denoted as the B19, R19, R33, R60, R65, R67, and R79 alleles of the dgs gene (Wolfgang, 2001).

Initial characterization of all alleles except dgsB19 indicated that each results in lethality at late embryonic or early first instar stages. To assess whether maternal Gsalpha contributes to embryonic survival, embryos from females with mutant germlines were examined. Ovaries in females carrying dgs germline mutations are apparently normal in all respects. For all alleles except dgsB19 and dgsR60, the percentage embryonic lethality increases in mothers with germline clones compared to zygotic mutants of the same genotype. However, in most cases (except dgsR19 homozygotes and all dgsB19 combinations), from 40% to 70% of the mutant embryos still hatch despite the absence of the germline as well as the zygotic dgs gene. The higher rate of embryonic lethality in dgsR19 homozygotes may be due to a second site recessive mutation carried on this chromosome since this was the only allelic combination that could not be rescued with the dgs transgene. As noted above, for all alleles except for dgsB19, mutant embryos that did hatch showed little movement, no growth, and died shortly after hatching. Similar to the results obtained in the case of zygotic mutant embryos, larvae that hatch from mothers with dgsB19 maternal clones die at varying times throughout all postembryonic stages. A few develop to pharate adults that never eclose. Finally, all maternally mutant embryos were rescued by a paternally contributed dgs gene to produce fertile adults (Wolfgang, 2001).

The control of cAMP synthesis by receptor regulation of adenylyl cyclase through GsalphaA is a primary mechanism for establishing intracellular cAMP concentrations. To determine the contribution of signaling through Gsalpha to the total level of cAMP in Drosophila, the effect of null (dgsR60 and dgsR79) and hypomorphic (dgsB19) dgs mutations on basal cAMP levels was determined in homozygous first instar larvae. In each case, the cAMP levels in mutant larvae were significantly lower than observed in controls. The mean cAMP concentration in Canton-S larvae was 59.3 ± 1.3 pmol/mg protein. Null dgs mutations reduce this level roughly four- to five-fold. In contrast, the hypomorphic dgsB19 mutation results in cAMP levels that are reduced ~40% when compared to control larvae. These results demonstrate that cellular levels of cAMP in Drosophila are determined in large part by signaling through Gsalpha (Wolfgang, 2001).

Embryos deficient in PKA show a variety of morphological defects, including alterations in cuticular patterning. To compare the effect of individual dgs mutations on embryonic pattern formation, cuticles were examined from late stage mutant embryos or early first instar larvae generated from germline clones, as well as those zygotically mutant. All dgs mutants that hatched had normal cuticle patterns. In addition, most dead embryos also had normal cuticle patterns, with the exception of dgsB19 mutant embryos generated from germline clones. In this case, of the mutant embryos that did not hatch, ~30% showed defects in the telson formation, 30% exhibited telson defects combined with posterior abdominal segment defects, and 30% were wild type. However, since only 4% of the total dgsB19/dgsR19 mutant population fail to hatch, the actual percentage of dgsB19 mutant embryos and first instar larvae showing patterning defects is only ~3%, similar to that observed for other alleles (1%-4% of the total mutant population). Thus, ~95%-99% of all mutant embryos show normal cuticular patterning (Wolfgang, 2001).

Observation of dgsB19 mutant larvae on egg plates and in bottles indicates that they have sluggish, uncoordinated movements. To quantify larval mobility and activity, the behavior of homozygous dgsB19 third instar larvae was assayed using the 'rover' assay. Individual larvae were placed on an egg plate uniformly coated with yeast and, after 5 min, the length of the track left in the yeast by the larvae was measured. These data were then represented as histograms showing the number of larvae (y-axis) that crawled a given distance (x-axis). Although there is some individual overlap, as a population, larvae homozygous or hemizygous for dgsB19 crawl shorter distances than heterozygous controls. There appears to be no dominant effect of the mutation on activity since dgsB19/CyO-GFP individuals show crawling activity similar to a variety of controls. The reduced crawling observed for dgsB19/dgsB19 homozygotes was rescued by introduction of the dgs transgene. During this analysis, it was noted that some dgsB19 mutant larvae often crawl in continuous circles, backward, or lie on their backs for extended periods of time, behaviors that are not observed in heterozygous or nonmutant larvae. It was also noted that dgsB19 mutants appear to not be attracted to yeast granules on the egg plates, suggesting sensory-motor deficits. These results indicate that dgsB19 mutant larvae have severe defects in neural and/or muscle physiology (Wolfgang, 2001).

The phenotypes generated by mutations in the dgs gene in Drosophila stand in direct contrast to those generated by mutations in the DC0 gene, encoding a presumed downstream effector of this pathway, PKA. For example, females carrying germline clones for DC0 mutations fail to lay eggs. In various heteroallelic combinations, PKA mutant females show defects in oogenesis due to the disruption of microtubule distribution and the localization of RNAs encoding key determinants (e.g., bicoid and oskar) along the anteroposterior axis. In contrast, adult females whose germlines carry dgs null alleles (e.g., dgsR60 and dgsR19) lay morphologically normal eggs that develop to late embryonic stages with ~50% hatching. Furthermore, embryos deficient in PKA also show a variety of morphological defects including preblastoderm arrest and alterations in cuticular patterning. Patterning defects arise due to the role of PKA as a modulator of the conversion of Cubitus interruptus (Ci), the transcription factor responsible for transducing signals mediated by the morphogen Hedgehog, from a transcriptional activator to a transcriptional repressor; in PKA-deficient embryos, Ci is not processed to the repressor form, resulting in phenotypes resembling ectopic hedgehog expression. In contrast, embryos maternally or zygotically deficient for Gsalpha do not show patterning defects consistent with alterations in the hedgehog signaling pathway. In addition, direct measurement of cAMP levels in larvae homozygous for dgs null mutations (dgsR60 and dgsR79) has shown that signaling through Gsalpha plays a major role in establishing basal levels of this second messenger. Since dgs mutations do not generate the embryonic patterning defects observed in DC0 mutants, basal PKA activity is likely not to depend on pathways activated by Gsalpha that contribute to basal levels of cAMP. Interestingly, alterations observed on a physiological level following mutational and pharmacological manipulation of cAMP levels cannot be mimicked, or are not observed to the same extent, following partial, mutational inactivation of PKA. These observations suggest that PKA activation by cAMP in Drosophila, at least in these developmental contexts, does not proceed by receptor-mediated modulation of the activity of adenylyl cyclase and increased intracellular cAMP. This interpretation is also consistent with two other observations: (1) phenotypes generated by expression of constitutively active forms of Gsalpha could not be suppressed by genetic and biochemical elimination of PKA activity; (2) expression of a cAMP-independent form of PKA in both embryos and imaginal discs is able to rescue phenotypes generated by elimination of PKA activity. These results are consistent with the conclusion that the activity of PKA, again at least in these developmental contexts, does not depend on receptor-mediated increases in intracellular cAMP, in contrast to the predictions of models developed primarily on the basis of studies in cultured cells. Since PKA is activated by cAMP, these observations leave open the question of how activation of PKA is mediated or of the role of cAMP in these contexts. However, these observations do not address the role of PKA in the generation of phenotypes present in dgs mutants (Wolfgang, 2001).

The results presented here further support the notion that Gsalpha-mediated activation of adenylyl cyclase in the production of cAMP and the activation of PKA are not necessarily coupled in a linear or dependent fashion. Three general alternatives can be invoked as the underlying basis for the differential response to the elimination of Gsalpha vs. PKA. (1) These genetic studies may point to the existence of a novel cAMP-independent signal transduction pathway activated directly by Gsalpha. Indeed, activation of cAMP-independent pathways by Gsalpha has been proposed in a variety of mammalian cell systems. (2) Alternatively, since expression of a constitutively activated form of Gsalpha in cultured mammalian cells results in increased intracellular cAMP, it may be that the primary mediator of the effects of cAMP in these cellular contexts is a molecule other than PKA, such as cyclic nucleotide-gated channels. In addition, (3) since gamma-subunits are potent modulators of a number of biochemical processes, it is possible that free ßgamma, generated as a consequence of the absence of Gsalpha, may in fact be responsible for some phenotypes associated with dgs mutations. Clearly, a goal of future studies will be to differentiate between these formal alternatives (Wolfgang, 2001).

Presynaptic impairment of synaptic transmission in Drosophila embryos lacking Gsalpha

Gsalpha is a subunit of the heterotrimeric G-protein complex, expressed ubiquitously in all types of cells, including neurons. Drosophila larvae, which have mutations in the Gsalpha gene, are lethargic, suggesting an impairment of neuronal functions. In this study, synaptic transmission was examined at the neuromuscular junction synapse in Gsalpha-null (dgsR60) embryos shortly before they hatched. At low-frequency nerve stimulation, synaptic transmission in mutant embryos was not very different from that in controls. In contrast, facilitation during tetanic stimulation was minimal in dgsR60, and no post-tetanic potentiation was observed. Miniature synaptic currents (mSCs) were slightly smaller in amplitude and less frequent in dgsR60 embryos in normal-K+ saline. In high-K+ saline, mSCs with distinctly large amplitude occurred frequently in controls at late embryonic stages, whereas those mSCs were rarely observed in dgsR60 embryos, suggesting a developmental defect in the mutant. Using the Gal4-UAS expression system, it was found that these phenotypes in dgsR60 were caused predominantly by lack of Gsalpha in presynaptic neurons and not in postsynaptic muscles. To test whether Gsalpha couples presynaptic modulator receptors to adenylyl cyclase (AC), the responses of two known G-protein-coupled receptors was examined in dgsR60 embryos. Both metabotropic glutamate and octopamine receptor responses were indistinguishable from those of controls, indicating that these receptors are not linked to AC by Gsalpha. It is therefore suggested that synaptic transmission is compromised in dgsR60 embryos because of presynaptic defects in two distinct processes; one is uncoupling between the yet-to-be-known modulator receptor and AC activation, and the other is a defect in synapse formation (Hou, 2003).

Two distinct sets of phenotypes in synaptic transmission at the neuromuscular synapse in dgsR60 embryos were revealed, as follows.
(1) Slightly smaller quantal content: The failure rate of stimuli to evoke synaptic currents in dgsR60 embryos was slightly greater in saline containing 0.2 mM Ca2+ compared with heterozygotes, suggesting smaller quantal contents of evoked synaptic currents. This subtle impairment in nerve-evoked synaptic transmission probably correlates with lower frequencies of mSCs. Synaptic impairment was more clearly demonstrated on stimulation at 10 Hz. In dgsR60 embryos, minimal synaptic facilitation was found during tetanus and no post-tetanic potentiation (PTP). Furthermore, asynchronous release of quanta during and after tetanus was much less than in controls. The lack of PTP found in dgsR60 embryos was similar to that in rut1. At the light-microscopic level, the morphology of neuromuscular synapses in dgsR60 embryos is not different from that of controls. Then the defects in dgsR60 embryos could be in a lower release probability, in a smaller number of release sites, or in a smaller number of release-ready vesicles.
(2) Smaller quantal size: Amplitudes of mSCs in dgsR60 embryos were slightly smaller in normal-K+ saline than in controls. The difference in mean miniature synaptic currents (mSC) amplitude was more clearly demonstrated in high-K+ saline, in which large mSCs occurred more frequently in controls than in dgsR60 embryos. Consequently, the amplitude histogram was broadly distributed in controls, whereas it was skewed toward large amplitudes in dgsR60. The frequent occurrence of large mSCs in high-K+ saline in controls may reflect a developmental process in synapse maturation, which might be defective in dgsR60 embryos. These two distinct sets of phenotypes in synaptic transmission in dgsR60 embryos are both a result of presynaptic defects (Hou, 2003).

In rut1, Ca2+-calmodulin-responsive AC is defective, and mGluR responses are markedly reduced. AC coded by rut therefore appears to at least partly mediate mGluR responses. If Gsalpha couples a modulator receptor to AC in nerve terminals, similar phenotypes could be expected in dgsR60 and in rut1 (Hou, 2003).

During tetanic stimulation, synaptic transmission was slightly facilitated in rut1 embryos and in dgsR60, but PTP was absent in both mutants. In third instars of a dgs hypomorph, dgsB19, both facilitation during tetanus and PTP were absent, and in third instars of rut1, there was slight facilitation during tetanus but no PTP. These phenotypes are similar between rut1 and dgs. However, the mean amplitude of mSCs in high-K+ saline was smaller in dgsR60 embryos than in rut1. The amplitude histogram was skewed in dgsR60 embryos, whereas in rut1 it was more widely distributed and indistinguishable from controls. Thus, between the two distinct sets of phenotypes in dgsR60 embryos, the slightly smaller quantal content is shared with rut1, but the smaller quantal size is not. It seems unlikely that the phenotypes in dgsR60 embryos result entirely from a mechanism similar to that in rut1, in which a low level of cAMP production during tetanus is probably underlying the lack of PTP (Hou, 2003).

Unexpectedly, in high-K+ saline many distinctly large mSCs were observed in Gs27 and dgsR60/+ embryos at the late embryonic stage. The mean amplitude was ~80% larger than in normal saline. The major factor contributing to these large mean amplitudes is frequent occurrence of large synaptic currents. Large mSCs do occur in normal-K+ saline, but their frequency is low. In high-K+ saline, their frequency was elevated disproportionately, resulting in the amplitude histograms with a broader and less-skewed distribution (Hou, 2003).

Two peaks in the mSC amplitude distribution have been reported in Xenopus nerve-muscle cultures. In younger cultures, the mean amplitude of mSCs is smaller and the amplitude distribution is skewed toward larger amplitudes. The second symmetrical peak in the large-amplitude range appears in older cultures. This change in the amplitude distribution is considered to be a developmental process. In Drosophila, a similar transition of amplitude histogram from a skewed distribution with a single peak to a distribution with two peaks during development has not been demonstrated. In this study, broader amplitude distributions and sometimes two peaks were observed in control strains in high-K+ saline. This could be a change in Drosophila embryos corresponding to that observed in Xenopus nerve-muscle cultures (Hou, 2003).

Why do large mSCs occur frequently in high-K+ saline? Among other possibilities, the following scenario is favored. In rapidly developing embryos, some release sites may be more mature than others. In high-K+ saline with Ca2+, in which Ca2+ levels in the presynaptic nerve terminal are elevated, fusion of vesicles may occur more frequently at those mature release sites than at immature sites. These mature release sites probably face a postsynaptic membrane with a higher receptor density. In dgsR60 embryos, however, fewer release sites may be mature and face a postsynaptic membrane with a high receptor density. In addition, those release sites may not be responding to an elevated Ca2+ in high-K+ saline to produce large mSCs, resulting in the smaller mean amplitude with a skewed distribution. In normal saline, these mature release sites may be regulated not to initiate excessive vesicle fusion. Because the mean amplitude of glutamate-induced currents reflecting the total number of receptors was not different between dgsR60 embryos and controls, these mature release sites with high receptor densities could not be more numerous in controls but must be releasing vesicles more frequently in high-K+ saline (Hou, 2003).

In this study, the skewed amplitude distributions of mSCs were found in dgsR60 embryos in high-K+ saline, whereas in controls, amplitude distributions were broader. Because a transition from a skewed amplitude distribution to a broader one has been observed during synapse formation, the skewed amplitude distributions in dgsR60 embryos could be an indication of immature synapses, suggesting the involvement of Gsalpha in synapse formation. An observation pointing to the involvement of Gsalpha in synapse formation was also made in third instar larvae of a hypomorphic mutant, dgsB19. The numbers of boutons and branches of presynaptic terminals at the neuromuscular synapse were smaller in dgsB19 third instars than in controls. These phenotypes were not observed in second instars of dgsB19. This finding suggests that during the period of rapid muscle expansion and synapse formation in third instars, activation of Gsalpha is required. The presynaptic defects in dgsR60 embryos may be related to a similar process during early synapse formation (Hou, 2003).

The effects of the null-mutation in dgs on synaptic transmission were observed in two aspects. One could be because of uncoupling between the as-yet-unknown modulator receptor and AC activation. This phenotype is similar to that in rut1. The other is probably a defect in synapse formation. Mature release sites with high receptor densities may not be well developed in dgsR60 embryos. To pinpoint the process in which Gsalpha is involved, it is necessary to further examine synaptic transmission at early stages of development (Hou, 2003).

Mutation and activation of Galphas similarly alters pre- and postsynaptic mechanisms modulating neurotransmission

Constitutive activation of Galphas in the Drosophila brain abolishes associative learning, a behavioral disruption far worse than that observed in any single cAMP metabolic mutant, suggesting that Galphas is essential for synaptic plasticity. The intent of this study was to examine the role of Galphas in regulating synaptic function by targeting constitutively active Galphas to either pre- or postsynaptic cells and by examining loss-of-function Galphas mutants (dgs) at the glutamatergic neuromuscular junction (NMJ) model synapse. Surprisingly, both loss of Galphas and activation of Galphas in either pre- or postsynaptic compartment similarly increases basal neurotransmission, decreases short-term plasticity (facilitation and augmentation), and abolished posttetanic potentiation. Elevated synaptic function is specific to an evoked neurotransmission pathway because both spontaneous synaptic vesicle fusion frequency and amplitude are unaltered in all mutants. In the postsynaptic cell, the glutamate receptor field is regulated by Galphas activity; based on immunocytochemical studies, GluRIIA receptor subunits are dramatically downregulated (>75% decrease) in both loss and constitutive active Galphas genotypes. In the presynaptic cell, the synaptic vesicle cycle is regulated by Galphas activity; based on FM1-43 dye imaging studies, evoked vesicle fusion rate is increased in both loss and constitutively active Galphas genotypes. An important conclusion of this study is that both increased and decreased Galphas activity very similarly alters pre- and post-synaptic mechanisms. A second important conclusion is that Galphas activity induces transynaptic signaling; targeted Galphas activation in the presynapse downregulates postsynaptic GluRIIA receptors, whereas targeted Galphas activation in the postsynapse enhances presynaptic vesicle cycling (Renden, 2003).

The objective of this study was to determine the role of the Galphas pathway in the regulation of synaptic transmission and functional plasticity and especially to assay synaptic correlates of the striking loss of behavioral learning observed following the constitutive activation of Galphas in the Drosophila brain. As with previous studies of plasticity mechanisms in Drosophila, this investigation made use of the larval, glutamatergic NMJ as the synaptic system for all assays. Surprisingly, both loss of Galphas (dgs mutants) and activation of Galphas (dgs* transgene) in either pre- or postsynaptic cells similarly results in a dramatic increase in evoked synaptic efficacy and concomitant loss of functional plasticity (facilitation, augmentation, and potentiation) in reduced [Ca2+]bath conditions. These results indicate that synaptic plasticity is dependent on proper Galphas-mediated signaling on both sides of the synapse. The behavioral learning deficit after dgs* expression is far worse than defects in cAMP metabolic mutants (dnc, rut, DC0), suggesting that cAMP-independent mechanisms are being misregulated via Galphas manipulation. Similarly, the NMJ transmission defects following dgs* expression are also more severe than those of cAMP metabolic mutants. It is concluded that the total scope of Galphas synaptic signaling, through cAMP and other pathways, is responsible for keeping the synapse within specific functional parameters to allow for rapid modification of transmission strength. Functionally, misregulation of Galphas activity affects the presynaptic vesicle cycle to change evoked pathway-specific changes in vesicle fusion probability. In addition, increased or decreased Galphas activity strikingly alters the composition of the postsynaptic glutamate receptor field, although the functional significance of this regulation is presently unclear. Finally, the level of Galphas activity is communicated transynaptically to bidirectionally control both pre- and post-synaptic mechanisms of neurotransmission (Renden, 2003).

The exaggerated excitatory junction current (EJC) basal neurotransmission and reduced short-term facilitation (STF) in reduced [Ca2+]bath conditions caused by Galphas activation is consistent with other conditions in which the cAMP-signaling pathway is activated. For example, the classic Drosophila plasticity mutant dnc (PDE; elevated cAMP) displays a twofold increase in basal EJC amplitude and reduced STF in 0.2 mM [Ca2+]. Similarly, the increased transmission caused by activated Galphas correlates with reduced Ca2+ dependence of transmission, similar to the reduction of Ca2+ dependence in dnc mutants. The observation that increased transmission caused by expression of dgs* is greater than that observed in dnc null mutants could point to opposing or compensatory roles of Galphas signaling, through cAMP or other intracellular cascades, in pre- versus post-synaptic compartments. Alternatively, the difference could be due to the simple fact that cAMP levels are more elevated in the dgs* GAL4-UAS manipulations than in the dnc mutants. Direct measurements of [cAMP] have been performed in both dnc mutants and in cells expressing the dgs* construct used in this study. In the null dnc alleles used in plasticity studies, cAMP was increased five- to six-fold over controls, and dnc hypomorphs showed a twofold increase in [cAMP]. dgs*-expressing cells show a much larger increase in basal [cAMP], reportedly 60-fold over controls (Renden, 2003).

Surprisingly, loss of Galphas activity (dgs mutants) shows similar trends in the elevation of basal transmission in low [Ca2+], altered Ca2+ dependence, and loss of short-term plasticity. Some, but not all, of these defects are also observed in the classic plasticity mutant rut (AC; decreased cAMP). When cAMP signaling is inhibited in rut mutants, basal transmission properties are not significantly altered, whereas short-term plasticity is decreased. However, loss of cAMP-dependent PKA activity, in genetic manipulations which overexpress the regulatory subunit of PKA, increase EJC amplitude nearly twofold (Renden, 2003).

PTP, measured in low (0.2 mM) [Ca2+], is essentially eliminated due to either activation or reduction of Galphas activity. This phenotype is also observed in the cAMP metabolic mutants, suggesting that Galphas-dependent regulation of cAMP levels is a central component required for the expression of synaptic potentiation. These results argue that Galphas signaling at the NMJ drives cAMP-dependent changes in synaptic efficacy but may involve pathways in parallel to the stimulation of the cAMP cascade, which account for the more significant, bidirectional alterations in transmission amplitude and activity-regulated plasticity (Renden, 2003).

Analysis of spontaneous vesicle fusion activity, in the absence of endogenous or exogenous excitation, is used routinely to dissect a pre- versus post-synaptic effects of a variety of mutants and pharmacological treatments at the Drosophila NMJ. In such quantal analyses, it is assumed that the packaging of neurotransmitter into vesicles is not altered or that the sensitivity or size of the receptor field is not affected in a compensatory manner. Thus a change in quantal fusion rate (mEJC frequency) reveals a presynaptic change (increased number of synapses, increased size of the readily releasable vesicle pool, increased probability of fusion), and a change in quantal size (mEJC amplitude) is indicative of a postsynaptic change (density of receptors, conductance state of receptors). Quantal analyses were therefore done to determine whether the dramatic 200%-400% increases in neurotransmission observed in gain and loss of Galphas mutants at low [Ca2+] was due to enhanced presynaptic release, postsynaptic response, or a combination of both (Renden, 2003).

Surprisingly, quantal analyses do not indicate enhancement of either pre- or post-synaptic function in Galphas mutants. The fact that there is no significant alteration in mEJC frequency in any condition activating or reducing Galphas activity suggests that the heightened transmission is not due to any change in spontaneous vesicle fusion rate (number of fusion sites or probability of fusion sites) but rather correlate with changes specific to evoked fusion (e.g., gating of ion channels, response to Ca2+ fusion trigger). Mutants defective in the cAMP pathway (dnc, rut) both result in a twofold decrease in mEJC frequency when measured at the single bouton level and a dramatic (3- to 4-fold) increase in mEJC frequency when measured in culture or in central neurons; however, mEJC frequency at the whole NMJ level has not been measured for these animals. The level of intracellular cAMP directly affects opening probability of shaker-type K+ channels, with dnc mutants showing a greater open probability, resulting in hyperexcitability and presumably altered mEJC frequency (Renden, 2003).

Similarly, mEJC amplitude is largely unaffected in Galphas mutants, suggesting little change in the density or conductance of postsynaptic glutamate receptors downstream of Galphas regulation. After postsynaptic activation of Galphas, a modest increase in quantal size (approximately 10%) was observed, but this increase is insufficient to explain the 400% increase in EJC amplitude (assuming that the relationship between spontaneous and evoked responses is linear). In dnc and rut mutants, mEJC amplitude is not altered. Thus an increase in basal glutamate receptor density or conductance properties does not appear to contribute significantly to the Galphas mutant phenotype (Renden, 2003).

It is important to note that similar enhancement in synaptic transmission amplitude and loss of functional plasticity, without any change in mEJC properties, has been reported for a number of other learning mutants in Drosophila. These findings suggest that alteration to evoked synaptic transmission mechanisms is the primary mechanism for regulating synaptic plasticity at the Drosophila NMJ. Numerous potential targets for this presynaptic regulation have been identified. In addition, activity-dependent changes in postsynaptic responsiveness are also possible. Fast glutamate receptor insertion and removal from postsynaptic membranes has been widely discussed recently as a mechanism for postsynaptic plasticity with the observation that AMPA receptors are quickly inserted into the postsynaptic membrane after tetanus. Assays to monitor spontaneous activity after tetanic stimulation indicate that this mechanism is unlikely to act at the Drosophila NMJ, at least in the 5-min time frame monitored in this study. Thus the results to date are consistent with an evoked pathway-specific alteration of presynaptic efficacy as the primary mechanism of plasticity downstream of both Galphas and cAMP (Renden, 2003).

The results presented here are consistent with a persistent state of presynaptic potentiation resulting when Galphas activity is either increased or decreased. FM 1-43-dye-labeling assays demonstrate that the rate of synaptic vesicle cycling through the exo-endo pool is significantly increased in low [Ca2+]bath conditions when Galphas is activated. Loss of function Galphas mutants show no significant difference in the size of the exo-endo pool. This is consistent with prior work, which has shown that the translocation from the reserve pool was selectively affected by PKA inhibitors. The more modest increase in transmission in these mutants (200%) must be restricted to altered evoked release of vesicles in the exo-endo pool only. Previous work in Drosophila has shown that cAMP-dependent regulation of Shaker K+ channels is likely to be a primary mechanism explaining elevated vesicle cycling. PKA phosphorylates K+ channels, and dnc and rut mutants interact with K+ channel mutants and differentially affect K+ channel conductance. A second likely mechanism involves Galphas-dependent increased Ca2+ influx. In mammalian neuronal cultures, cAMP has been shown to increase N- and L-type Ca2+ channel currents, in a PKA-dependent manner, and these same Ca2+ currents are altered proportionally by dnc, rut, or effectors or inhibitors of the cAMP pathway. Such a mechanism would explain the altered Ca2+ dependence of neurotransmitter release observed in Galphas mutants. A third possible mechanism is directly increased vesicle mobilization, resulting in an activity-dependent alteration in the distribution of vesicles. Increased [cAMP] directly affects the mobility of the readily releasable synaptic vesicle pool at the Drosophila NMJ, making it more accessible to stimulated release. One or more of these mechanisms would explain the heightened low-Ca2+-evoked neurotransmission in Galphas gain-of-function mutants in the absence of any change in mEJC characteristics (Renden, 2003).

This study provides no evidence that postsynaptic function is regulated by the level of Galphas activity or that alterations in the postsynaptic glutamate receptor field play any role in short-term plasticity at the Drosophila NMJ. In both gain- and loss-of-function Galphas mutants, there is no substantial change in glutamate receptor conductance, density, or distribution based on mEJC amplitude analyses and direct assay of glutamate-gated currents in the muscle. This finding is extremely surprising in light of the dramatic alteration of the molecular character of the postsynaptic glutamate receptor field in both loss and gain of function Galphas mutants. Two different antibodies were used to assay the GluR fields: a polyclonal antibody against all GluRII subunits showed a significant reduction of signal in all Galphas mutants and a monoclonal antibody specific to GluRIIA showed a nearly complete loss of signal in all Galphas mutants. Immunoreactivity against DGluRIIA in the embryo appeared normal in dgsR60 homozygous mutants, indicating a postembryonic modification of DGluRIIA expression under the control of dgs. At a minimum, these analyses reveal a striking molecular alteration of the GluR field downstream of Galphas, possibly to the extent of nearly eliminating GluRIIA subunits (Renden, 2003).

Complete loss of GluRIIA has been shown to cause significantly decreased mEJC amplitudes, whereas a nearly complete loss of GluRIIA immunoreactivity, using two antibodies, is reported here without a similar change in mEJC amplitudes. One way to rationalize this apparent contradiction is to postulate that the reduced presence of GluRIIA after Galphas manipulation is not sufficient to alter significantly mEJC kinetics or amplitudes. The present report shows a 75% reduction of receptor abundance, whereas GluRIIA genetic nulls were examined previously. More recently, the effect of graded expression levels of GluRIIA was examined, revealing that low levels of GluRIIA, in the absence of GluRIIB, results in an overcompensation of presynaptic transmitter release, doubling the amplitude of glutamatergic transmission at the NMJ. At higher levels of GluRIIA expression, this phenotype was eliminated. If the levels of DGluRIIB were also downregulated (or eliminated) by altered Galphas signaling, these findings would be in agreement with those of the previous study. A second possibility is that the loss of GluRIIA immunoreactivity caused by Galphas manipulation may represent epitope masking rather than loss of GluRIIA subunits. Extracellular binding of an auxiliary protein to glutamate receptors has recently been reported in C. elegans, and an essential auxiliary subunit of mammalian AMPA receptors (stargazin) has recently been found. Stargazin is essential for proper insertion and localization of receptors with the postsynaptic density and is modulated by PKA phosphorylation, thereby controlling receptor number. Interaction with such proteins, or other changes in the accessibility/confirmation of GluRIIA in the postsynaptic compartment, might alter its recognition by antibodies. A final possibility is that there may be compensatory increases in the levels of the other GluRII subunits present at the NMJ. Such a compensatory mechanism might permit loss of GluRIIA subunits without an appreciable change in mEJC amplitudes. The loss of GluRIIA immunoreactivity demonstrates conclusively that the postsynaptic GluR field is strikingly controlled by the level of Galphas activity, but the functional significance of this regulation remains elusive and awaits further investigation (Renden, 2003).

Work on Gs-mediated plasticity has focused almost entirely on the cAMP cascade regulated by the alpha subunit. However, activation of Gs results in the dissociation of the heterotrimeric complex into free alpha subunits and betagamma complexes, and each mediates distinctive intracellular signaling. Relatively little work has been published specifically on the effects of betagamma signaling due to Gs-coupled receptor activation, but it is believed that the betagamma subunits are not very selective and can be shared between all G proteins. Recent work has shown that the addition of betagamma increases L-type channel Ca2+ influx via a PKC-mediated pathway and directly mediates activation of K+Ach current due to receptor activation of Gs. If these two results are extrapolated to the Drosophila NMJ, transgenic manipulation of Gs signaling could give rise to alterations in Ca2+ and K+ currents independent of Galphas activity. betagamma subunits have been shown to decrease P/Q and N-type Ca2+ channels by binding them directly at the I-II intracellular loop. Classical second-messenger pathways are also modulated by betagamma signaling: type-specific inhibition or activation of adenylyl cyclase (AC1and AC2, respectively) as well as activation of phospholipase Cbeta. In addition, recent work has shown that other receptor kinases, such as ras/MAP kinase, can be activated specifically by betagamma (Renden, 2003).

It is not clear how free betagamma subunits are regulated by the cell, although there is evidence that they are physically sequestered by tethering proteins such as phosducin. It is possible that excess betagamma complexes are present when Galphas is constitutively activated and that they could effect a wide variety of intracellular signaling cascades, leading in part to the novel physiological phenotypes seen in this study. However, there are few examples where endogenous betagamma signaling predominates the modulation of intracellular signaling (Renden, 2003).

Numerous lines of evidence have demonstrated the existence of both anterograde and retrograde transynaptic signals at the Drosophila NMJ. Such signals are involved in induction of postsynaptic receptor fields, pruning of postsynaptic receptor fields, and compensatory regulation of presynaptic quantal content. The present study shows that increasing Galphas function either pre- or postsynaptically results in nearly identical phenotypes, and independent assays of presynaptic and postsynaptic function indicate similar mechanisms. Specifically, presynaptic Galphas activation modifies the postsynaptic GluRIIA receptor field, and postsynaptic Galphas activation heightens presynaptic vesicle cycling. Moreover, global loss-of-function Galphas mutants also modify the postsynaptic GluRIIA field. Are these paired pre- and post-synaptic alterations a form of compensation or are they independent, Galphas-dependent mechanisms? What signals are used to communicate the level of Galphas activity in both directions across the synaptic cleft (Renden, 2003)?

The identity of the messenger(s) is still unclear, but there are a few likely suspects. Glutamate itself has been shown to act as an anterograde regulatory message at the Drosophila NMJ; presynaptic glutamatergic tone inversely controls the levels of DGluRIIA postsynaptically. Thus it is possible that the elevated glutamatergic transmission in Galphas mutants directly causes the downregulation of GluRIIA expression. At the Drosophila NMJ and in mammalian systems, integrin function has been shown to be required for functional synaptic plasticity. Integrins are known to signal between cells within a short period of time through activation of associated intracellular cascades. At the Drosophila NMJ, the hypertonicity response is mediated in part by integrins dependent on intracellular cAMP levels, and in Xenopus cultured neurons, PKA-dependent transmission is inhibited by disintegrin. These studies suggest that integrins may function as anterograde and/or retrograde messengers mediating physical transynaptic signaling. Another possible retrograde messenger is nitric oxide, produced by phosphorylation of nitric oxide synthase (NOS). There is evidence that NOS is present in Drosophila and is localized to epithelial and neuronal tissues . Application of nitric oxide to the NMJ induces presynaptic vesicle fusion, making it a formal candidate as a retrograde messenger (Renden, 2003).

In conclusion, tissue-specific expression of constitutively active Galphas on either side of the Drosophila NMJ synaptic cleft greatly enhances basal neurotransmission to disrupt expression of short-term synaptic plasticity, specifically in low [Ca2+]bath conditions. This Galphas-dependent alteration does not affect the probability of spontaneous vesicle fusion or the basal function of the postsynaptic receptor field and so is specific to evoked release of neurotransmitter. Increases in Galphas activity on either side of the synapse greatly increases evoked amplitude in low Ca2+, primarily due to a cAMP-dependent increased synaptic vesicle mobility, but also dramatically reduce GluRIIA receptor levels. When Galphas activity is decreased, neurotransmission is similarly enhanced, GluRIIA receptor levels are similarly downregulated, but synaptic vesicle mobility is not detectably altered. It is clear that there is a bidirectional transynaptic communication network at the Drosophila NMJ that responds to altered Galphas activity to modify both pre- and post-synaptic compartments. However, the functional significance of some of these changes remains unclear, and the messengers mediating transynaptic signaling remain to be identified (Renden, 2003).

Dual roles for the trimeric G protein Go in asymmetric cell division in Drosophila

During asymmetric division, a cell polarizes and differentially distributes components to its opposite ends. The subsequent division differentially segregates the two component pools to the daughters, which thereby inherit different developmental directives. In Drosophila sensory organ precursor cells, the localization of Numb protein to the cell's anterior cortex is a key patterning event and is achieved by the combined action of many proteins, including Pins, which itself is localized anteriorly. This study describes a role for the trimeric G protein Go in the anterior localization of Numb and daughter cell fate specification. Go is shown to interact with Pins. In addition to a role in recruiting Numb to an asymmetric location in the cell's cortex, Go transduces a signal from the Frizzled receptor that directs the position in which the complex forms. Thus, Go likely integrates the signaling that directs the formation of the complex with the signaling that directs where the complex forms (Katanaev, 2006 see full text of article).

Because Fz appears to act as the exchange factor for Go in the Wnt and PCP pathways (Katanaev, 1995), The effects of GoWT and GoGTP on wing margin bristles were examined when Fz levels were modulated. The effects of overexpression of GoWT fell to zero in fz–/– wings, but the GoGTP overexpression phenotypes were not reduced; rather, they were enhanced. Why the aberrations increased is not clear, but this result shows that GoGTP is a potent disturber of asymmetric division in the absence of Fz, whereas WT Go requires it. This finding suggests that Go requires Fz to convert it into the 'active' GTP-bound state and predicts that overexpression of Fz should enhance the potency of Go. Indeed, co-overexpression of Fz and GoWT enhances the asymmetric division defects. Overexpression of Fz alone produced orientation defects but no asymmetric division aberrations (Katanaev, 2006).

In Drosophila, Wnt-1 (Wingless, Wg) is transduced by the Go-dependent receptors Fz and Dfz2. Therefore whether co-overexpression of Dfz2 could also enhance the effects of overexpression of Go was tested. Overexpression of Dfz2 alone characteristically induced ectopic margin bristles (activation of the Wg pathway) that showed no asymmetric division defects. But when Dfz2 and GoWT were co-overexpressed, they mutually enhanced their respective phenotypes, suggesting that Go enhanced the ability of Dfz2 to ectopically activate Wg signaling, and Dfz2 potentiated the ability of Go to disturb the asymmetric divisions. Dfz2 is usually down-regulated in the SOP region of the wing margin and likely does not normally influence Go activity there, but its forced expression shows an ability to potentiate the effects of Go (by inference catalyzing it into the GTP-activated form). These results provide the first example of the ability of Dfz2 to activate signaling in a pathway other than 'canonical' Wnt cascade (Katanaev, 2006).

Gβ13F and Gγ1 likely represent the β- and γ-subunits of the Go trimeric complex. Receptor-catalyzed exchange of GDP for GTP occurs on Gα-subunits complexed with βγ. Thus, βγ-subunits should be required for the effects of GoWT overexpression. Indeed, GoWT overexpression effects were attenuated when one gene copy of Gγ1 was removed, arguing that these effects were not due to sequestration of βγ moieties from another α-subunit such as Gi. Ablation of Gβ13F or Gγ1 genes was reported to affect neuroblast divisions. It was also found that loss or overexpression of Gγ1 and Gβ13F (but not Gβ5) resulted in adult bristle defects similar to those of loss or overexpression of Go. Taken together, these observations suggest that Gβ13F and Gγ1 represent the β- and γ-subunits of the Go trimeric complex (Katanaev, 2006).

Various roles for trimeric G proteins have been reported for asymmetric cell divisions; for example, Caenorhabditis elegans Gα-subunits GOA-1 and GPA-16 redundantly regulate posterior displacement of the mitotic spindle required for the asymmetric division of the zygote, and β- and γ-subunits are involved in orientating the mitotic spindle. In Drosophila, evidence for trimeric G protein function in both the formation of the asymmetric spindle and the correct localization of various cell fate determinants have come from manipulation of βγ-subunits in the neuroblasts. Additionally, Gi is known to be involved in asymmetric divisions and to interact with Pins; cell fate determinant localizations are aberrant during metaphase but are restored by telophase (Katanaev, 2006).

In this report, strong and pervasive roles have been documented for Go in Drosophila asymmetric divisions. Five major points are made: (1) In SOP asymmetric divisions, there are two patterning mechanisms: the establishment of the asymmetric complexes and the orientation of the asymmetry. Go appears to act in both functions and is therefore a likely molecular integrator of the two. (2) Go appears to function in both the neuroblast-type and SOP divisions and is therefore likely used in all asymmetric divisions in Drosophila. (3) Go binds to and genetically interacts with Pins. One function of Go, then, is likely mediated by a direct interaction with Pins. (4) Hitherto, Gi was considered the major Gα-subunit functioning in asymmetric cell divisions. Go shows significantly stronger phenotypes, suggesting a greater role, but genetic interaction between the two suggests a degree of functional redundancy. (5) Both Fz and Dfz2 appear able to act as exchange factors for Go in the SOP divisions. The role for Fz is supported by many different results, but whether Dfz2 normally functions here remains unclear (Katanaev, 2006).

Go appears to play parallel bifunctional roles in the establishment of asymmetries in both SOPs and PCP, as evidenced by the following: (1) polarized structures form in both; in PCP, it is the focal organizer of hair outgrowth, and in SOPs, it is the Numb crescent; (2) in both processes, Fz signaling organizes the polarized distribution of 'core group' PCP proteins. For example, Fz itself becomes localized to the distal and posterior ends of PCP cells and SOPs, respectively, whereas Van Gogh/Strabismus is found proximal and anterior in PCP cells and SOPs, respectively. (3) In both processes, these Fz-dependent localizations do not critically contribute to the final polarized structures, because loss of Fz (or other core group proteins) only leads to randomization in the positioning of the (usually) single-hair focus or Numb complex. Thus, there appear to be two semiindependent mechanisms: (1) the polarization of the core group PCP proteins, which instructs (2) the position of the self-assembling complexes (Katanaev, 2006).

Go appears to work in both these mechanisms. Mildly Go-compromised cells lose correct orientation of hairs or Numb complexes, consistent with an orientation function. Cells with strongly disturbed Go function lose the ability to polarize; in the SOP, Numb becomes diffuse or forms a number of small foci; and in PCP, many hair initiation sites are produced. Phenotypes of fz or other core group mutants occasionally result in two hairs per cell, but Go mutants frequently induce cells with five or six hairs (Katanaev, 2006).

The question now arises as to whether Go functions in the same way in both processes. In terms of the Fz-mediated orientation step, it is likely that Go performs the same role; in both, Fz is directed to one end of the cell (distal or posterior), and Go itself becomes preferentially distributed to the other end (proximal or anterior). This local enrichment of Go possibly serves as the point of integration with the internal asymmetry formation step. In the SOP case, anterior Go may recruit Pins and seed the formation of the anterior Numb crescent. In the PCP case, Go localizes opposite to the site of hair growth, suggesting that the highest depletion of Go specifies the site of hair growth. In the absence of the Fz orienting information, it may be a stochastic increase of Go localization (or activity) that establishes the initial asymmetric bias. Alternatively, the asymmetric distribution of Go may only be a manifestation of the Fz-mediated orientation, being essentially irrelevant to the subsequent step. In this case, the activity of Go (rather than its site of accumulation) would be required for the formation of the Numb crescent or the hair initiation point (Katanaev, 2006).

Competing activities of heterotrimeric G proteins in Drosophila wing maturation

Drosophila genome encodes six α-subunits of heterotrimeric G proteins. The α-subunit termed Gαs is involved in the post-eclosion wing maturation, which consists of the epithelial-mesenchymal transition and cell death, accompanied by unfolding of the pupal wing into the firm adult flight organ. This study shows that another α-subunit, Gαo, can specifically antagonize the Gαs activities by competing for the Gβ13F/Ggamma1 subunits of the heterotrimeric Gs protein complex. Loss of Gβ13F, Gγ1, or Gαs, but not any other G protein subunit, results in prevention of post-eclosion cell death and failure of the wing expansion. However, cell death prevention alone is not sufficient to induce the expansion defect, suggesting that the failure of epithelial-mesenchymal transition is key to the folded wing phenotypes. Overactivation of Gαs with cholera toxin mimics expression of constitutively activated Gαs and promotes wing blistering due to precocious cell death. In contrast, co-overexpression of Gβ13F and Gγ1 does not produce wing blistering, revealing the passive role of the Gβγ in the Gαs-mediated activation of apoptosis, but hinting at the possible function of Gβγ in the epithelial-mesenchymal transition. These results provide a comprehensive functional analysis of the heterotrimeric G protein proteome in the late stages of Drosophila wing development (Katanayeva, 2010).

G protein-coupled receptors (GPCRs) represent the most populous receptor family in metazoans. Approximately 380 non-olfactory GPCRs are encoded by the human genome, corroborated by ca. 250 GPCRs in insect genomes, making 1%-1.5% of the total gene number dedicated to this receptor superfamily in invertebrates and mammals. GPCRs transmit their signals by activating heterotrimeric G protein complexes inside the cell. A heterotrimeric G protein consists of a GDP-bound α-subunit and a βα-heterodimer. Ligand-stimulated GPCR serves as a guanine nucleotide-exchange factor, activating the GDP-to-GTP exchange on the Gα-subunit. This leads to dissociation of the heterotrimeric complex into Gα-GTP and flγ, which transmit the signal further inside the cell (Katanayeva, 2010).

The β- and γ-subunit repertoire of the Drosophila genome is reduced as compared with that of mammals: only two Gγ and three Gβ genes are present in flies. Gγ30A and Gβ76C are components of the fly phototransduction cascade and are mostly expressed in the visual system. Gγ1 and Gβ13F have been implicated in the asymmetric cell divisions and gastrulation, while the function of Gβ5 is as yet unknown (Katanayeva, 2010).

Despite the fact that βγ can activate signal effectors, the main selectivity in GPCR coupling and effector activation is provided by the Gα-subunits. Sixteen genes for the α-subunits are present in the human genome, and six in Drosophila. All human Gαsubunit subgroups are represented in Drosophila: Gαi and Gαo belonging to the Gαi/o subgroup; Gαq belonging to the Gαq/11 subgroup; Gαs belonging to the Gαs subgroup, and concertina (cta) belonging to the Gα12/13 subgroup. Additionally, Drosophila genome encodes for Gαf which probably represents an insect-specific subfamily of Gαsubunits (Katanayeva, 2010).

Multiple functions have been allocated to different heterotrimeric G proteins in humans and flies. For example, in Drosophila development cta is a crucial gastrulation regulator, Gαo is important for the transduction of the Wnt/Frizzled signaling cascade, and Gαi controls asymmetric cell divisions during generation of the central and peripheral nervous system (the later in cooperation with Gαo. Gαq is the Drosophila phototransduction Gαsubunit, but probably has additional functions. Pleotropic effects arise from defects in Gαs function, while the function of Gαf has not yet been characterized (Katanayeva, 2010).

Among the developmental processes ascribed to the control by Gαs are the latest stages of Drosophila wing development. Newly hatched flies have soft and folded wings, which during the 1-2 hours post-eclosion expand and harden through intensive synthesis of components of the extracellular matrix. These processes are accompanied by epithelial-mesenchymal transition and apoptosis of the wing epithelial cells, producing a strong but mostly dead adult wing structure. Expression of the constitutively active form of Gαs leads to precocious cell death in the wing epidermis, which results in failure of the closure of the dorsal and ventral wing sheets and accumulation of the hemolymph inside the wing, producing wing blistering. Conversely, clonal elimination of Gαs leads to autonomous prevention of the cell death. Kimura (2004) has performed an extensive analysis of the signaling pathway controlling apoptosis at late stages of wing development. That study provided evidence suggesting that the hormone bursicon, synthesized in the head of post-eclosion Drosophila and secreted in the hemolymph, activates a GPCR Rickets on wing epithelial cells, which signals through Gαs to activate the cAMP-PKA pathway, culminating at the induction of apoptosis. However, the identity and importance of the &βγ subunits in bursicon signaling, as well as possible involvement of other Ga proteins remained outside of their investigation. There also remain some uncertainties as to the phenotypic consequences of elimination of the bursicon-Gαs-PKA pathway in wings (Katanayeva, 2010).

This study describes a comprehensive functional analysis of the Drosophila heterotrimeric G protein proteome using loss-of-function and overexpression experiments. Loss of Gαs but not any other Gαsubunit leads to the failure of wing expansion after fly hatching. Gαo, but not another Gα, can compete with Gαs and thus antagonize its function. Finally, the Gβ13F and Gγ1 as the βγ subunits of the heterotrimeric Gs complex responding to the epithelial-mesenchymal transition and cell death-promoting signal (Katanayeva, 2010).

The soft folded wings of the young insect freshly hatched from the pupal case within 1-2 hours expand and harden, becoming a robust flight organ. This process is accompanied by epithelial-mesenchymal transition and cell death of the wing epithelial cells. Genetic dissection has revealed the function of the neurohormone bursicon and its wing epithelial receptor rickets in initiation of these processes. The GPCR rickets couples to the heterotrimeric G protein Gs; the Gαs-activated cAMP-PKA pathway culminates at the induction of apoptosis. However, the overall phenotypic consequences of the loss of the Gs signaling pathway in post-eclosion wings were unknown, as well as the nature of the Gβγ subunits of the heterotrimeric Gs complex responding to the bursicon-rickets signaling (Katanayeva, 2010).

This study consisted of an extensive analysis of the heterotrimeric G protein subunits in these post-eclosion stages of wing maturation. The whole-wing down-regulation of Gαs results in the failure of wing expansion, demonstrating that this change in the shape of the wing is the major morphological outcome of the bursicon-rickets-Gs signaling. The Gβ13F and Gγ1 subunits were also identified as the other two constituents of the heterotrimeric Gs complex, as downregulation of Gαs, Gβ13F, or Gγ1, but not any other Ga, Gβ, or Gγ subunits encoded by the Drosophila genome, each leads to the same folded wing phenotype (Katanayeva, 2010).

It was also shown that Gαo, but not any other Gαsubunit, can inhibit the wing expansion program through sequestration of the Gβ13F/Gγ1 heterodimer. The reason for the specificity of Gαo over other Gαsubunits in antagonizing the Gs signaling is unclear. It is unlikely that differences in expression levels of the tested Gαsubunits may account for the selective activity of Gαo. Indeed, most overexpression experiments were done with the X-chromosome-inserted MS1096-Gal4 driver, which results in markedly higher expression levels in males than heterozygous female flies, producing a more penetrant folded wing phenotype in males overexpressing Gαo. However, even in male flies overexpressing other Gαsubunits no instances of the folded wing phenotype could be seen. Furthermore, several independent insertions of the UAS-Ga transgenes were tested; while different Gαo transgenes all produced the folded wing phenotype upon overexpression, other Ga constructs remained ineffective (Katanayeva, 2010).

Similarly, the different Gαsubunits possess a similar affinity towards the interaction with the Gβγ heterodimer, not providing an explanation for a specific ability of Gαo to antagonize the Gs-mediated post-eclosion pathway. It is thus thus tempting to propose that a previously uncharacterized biochemical mechanism may allow for a specific antagonism physiologically existing between the Gs- and Go- mediated signaling pathways. As liberation of high amounts of GDP-loaded Gαo is predicted to be a consequence of activation of multiple Go-coupled GPCRs, and as Go is a heavily expressed G protein representing the major G protein species e.g. in the brain of flies and mammals, this specific ability of Gαo to antagonize the Gs-mediated signaling may have physiological implications in other tissues and organisms than Drosophila wing. However, it is added that these speculations are based on the analysis of the overexpression data and must be treated with caution when translating them into physiological situations (Katanayeva, 2010).

Only the GDP-loaded, but not the activated GTP-loaded form of Gαo is effective in antagonizing Gs. A proteomics analysis was performed of the Drosophila proteins which would discriminate between the two nucleotide forms of Gαo, and surprisingly few targets of this kind were revealed. While the chaperone Hsc70-3 and β1-tubulin preferentially interacted with the GTP-loaded Gαo, Gβ13F was found to specifically interact with Gαo-GDP. These data suggest that many Gαo-interaction partners do not discriminate between the two guanine forms of Gαo. These findings are in agreement with our other experimental findings, as well as mathematical modeling predicting that high concentrations of free (monomeric) signaling-competent Gαo-GDP are produced upon activation of Go-coupled GPCRs (Katanayeva, 2010).

Gαo-mediated sequestration of Gβ13F/Gγ1 depletes the pool of the heterotrimeric Gs complexes. As only heterotrimeric Ga&βγ, but not monomeric Ga proteins can efficiently bind and be activated by their cognate GPCRs, overexpression of Gαo abrogates the rickets-Gs signaling. Phenotypic consequences of this abrogation are the failures of apoptosis and wing expansion. In contrast, expression of the constitutively activated form of Gαs induces premature cell death and wing blistering. This phenotype can be also induced by expression of cholera toxin, revealing that the ability of cholera toxin to specifically overactivate Gαs reported in mammalian systems is reproduced with Drosophila proteins. These data also confirm that not only exogenously overexpressed, but also the endogenous Gαs can induce the precocious cell death upon overactivation (Katanayeva, 2010).

However, prevention of apoptosis is not sufficient to produce the folded wing phenotype. Together with the observation that the constitutively active form of Gαs is ineffective in rescuing the wing expansion defects produced by Gαo overexpression, these data suggest that the Gαs-cAMP-PKA pathway culminating at apoptosis is not the sole signaling branch emanating from the bursicon-rickets GPCR activation. It is proposed that the second signaling branch initiated by the rickets-mediated dissociation of the heterotrimeric Gs complex is represented by the free Gββ subunits, signaling to epithelial-mesenchymal transition. Such a double signaling impact mediated by the two components of the heterotrimeric G protein complex leads to initiation of two cellular programs -- apoptosis and epithelial-mesenchymal transition -- which cumulatively result in wing expansion and solidification, producing the adult flight organ. This two-fold response of the Drosophila wing to the maturation signal, mediated by the two components of the heterotrimeric G protein complex activated by the single hormone-responsive GPCR, provides an elegant paradigm for the coordination of signaling and developmental programs (Katanayeva, 2010).


Bejarano, F., Luque, C. M., Herranz, H., Sorrosal, G., Rafel, N., et al., (2008). A gain-of-function suppressor screen for genes involved in dorsal-ventral boundary formation in the Drosophila wing. Genetics 178: 307-323. PubMed Citation: 18202376

Brembs, B. and Heisenberg, M (2000). The operant and the classical in conditioned orientation of Drosophila melanogaster at the flight simulator. Learn. Mem. 7: 104-115. 10753977

Bronstein, R., et al. (2011a). Transcriptional regulation by CHIP/LDB complexes. PLoS Genet. 6(8): e1001063. PubMed Citation: 20730086

Cagampang, F. R., et al. (1998). Circadian changes of type II adenylyl cyclase mRNA in the rat suprachiasmatic nuclei. Brain Res. 810(1-2): 279-82. PubMed Citation: 9813369

Chakrabarti, S., et al. (1998). Chronic morphine augments G(beta)(gamma)/Gs(alpha) stimulation of adenylyl cyclase: relevance to opioid tolerance. Mol. Pharmacol. 54(4): 655-62. PubMed Citation: 9765508

Chen, L., et al. (2002). Ssdp proteins interact with the LIM-domain-binding protein Ldb1 to regulate development. Proc. Natl. Acad. Sci. 99: 14320-14325. PubMed Citation: 12381786

Chen, X. and Ganetzky, B. (2012). A neuropeptide signaling pathway regulates synaptic growth in Drosophila. J Cell Biol 196: 529-543. PubMed ID: 22331845

Connolly, J. B., et al. (1996), Associative learning disrupted by impaired Gs signaling in Drosophila mushroom bodies. Science 274: 2104-2107. PubMed Citation: 8953046

Costa, M., Wilson, E. T. and Wieschaus, E. (1994). A putative cell signal encoded by the folded gastrulation gene coordinates cell shape changes during Drosophila gastrulation. Cell 76: 1075-89. PubMed Citation: 8137424

Dahdal, D., et al. (2010). Drosophila pacemaker neurons require g protein signaling and GABAergic inputs to generate twenty-four hour behavioral rhythms. Neuron 68(5): 964-77. PubMed Citation: 21145008

Ferris, J., Ge, H., Liu, L. and Roman, G. (2006). G(o) signaling is required for Drosophila associative learning. Nat. Neurosci. 9(8): 1036-40. 16845387

Fromm, C., et al. (1997). The small GTP-binding protein Rho links G protein-coupled receptors and Galpha12 to the serum response element and to cellular transformation. Proc. Natl. Acad. Sci. 94(19): 10098-10103. PubMed Citation: 9294169

Girgenrath, S. and Smith, W. A. (1996). Investigation of presumptive mobilization pathways for calcium in the steroidogenic action of big prothoracicotropic hormone. Insect Biochem. Mol. Biol. 26(5): 455-463. PubMed Citation: 8763164

Govindan, J. A., et al. (2009). Somatic cAMP signaling regulates MSP-dependent oocyte growth and meiotic maturation in C. elegans. Development 136(13): 2211-21. PubMed Citation: 19502483

Hamm, H. E. (1998). The many faces of G protein signaling. J. Biol. Chem. 273: 669-672. PubMed Citation: 9422713

Hayward, B. E., et al. (1998). The human GNAS1 gene is imprinted and encodes distinct paternally and biallelically expressed G proteins. Proc. Natl. Acad. Sci. 95(17): 10038-43. PubMed Citation: 9707596

Hou, D., et al. (2003). Presynaptic impairment of synaptic transmission in Drosophila embryos lacking Gsalpha. J. Neurosci. 23(13): 5897-5905. 12843294

Huang, L. J., et al. (1997). D-AKAP2, a novel protein kinase A anchoring protein with a putative RGS domain. Proc. Natl. Acad. Sci. 94(21): 11184-11189. PubMed Citation: 9326583

Huang, Y. Y. and Kandel, E. R. (1995). D1/D5 receptor agonists induce a protein synthesis-dependent late potentiation in the CA1 region of the hippocampus. Proc. Natl. Acad. Sci. 92: 2446-50. PubMed Citation: 7708662

Iiri, T., et al. (1999). A Gsalpha mutant designed to inhibit receptor signaling through Gs. Proc. Natl. Acad. Sci. 96(2): 499-504. PubMed Citation: 9892662

Jho, E. H., Davis, R. J. and Malbon, C. C. (1997). c-Jun amino-terminal kinase is regulated by Galpha12/Galpha13 and obligate for differentiation of P19 embryonal carcinoma cells by retinoic acid. J. Biol. Chem. 272(39): 24468-24474. PubMed Citation: 9305908

Katanaev, V. L., Ponzielli, R., Semeriva, M. and Tomlinson, A. (2005). Trimeric G protein-dependent Frizzled signaling in Drosophila. Cell 120: 111-122. 15652486

Katanaev, V. L., Tomlinson, A. (2006). Dual roles for the trimeric G protein Go in asymmetric cell division in Drosophila. Proc. Natl. Acad. Sci. 103: 6524-6529. 16617104

Katanayeva, N., Kopein, D., Portmann, R., Hess, D. and Katanaev, V. L. (2010). Competing activities of heterotrimeric G proteins in Drosophila wing maturation. PLoS One. 5(8): e12331. PubMed Citation: 20808795

Kula-Eversole, E., Nagoshi, E., Shang, Y., Rodriguez, J., Allada, R. and Rosbash, M. (2010). Surprising gene expression patterns within and between PDF-containing circadian neurons in Drosophila. Proc. Natl. Acad. Sci. 107: 13497-502. PubMed Citation: 20624977

Kume, S., Inoue, T. and Mikoshiba, K. (2000). Galphas family G proteins activate IP3-Ca2+ signaling via Gbetagammma and transduce ventralizing signals in Xenopus. Dev. Biol. 226: 88-103. PubMed Citation: 10993676

Lader, A. S., et al. (1998). Cardiac gsalpha overexpression enhances L-type calcium channels through an adenylyl cyclase independent pathway. Proc. Natl. Acad. Sci. 95(16): 9669-9674. PubMed Citation: 9689139

Lans, H., Rademakers, S. and Jansen, G. (2004). A network of stimulatory and inhibitory Galpha-subunits regulates olfaction in Caenorhabditis elegans. PubMed Citation: 15342507. Genetics 167(4): 1677-87. 15342507

Liu, G., Seiler, H., Wen, A., Zars, T., Ito, K., Wolf, R., Heisenberg, M. and Liu, L. (2006). Distinct memory traces for two visual features in the Drosophila brain. Nature 439(7076): 551-6. 16452971

Ma. Y.-C. et al. (2000). Src tyrosine kinase is a novel direct effector of G proteins. Cell 102: 635-646. PubMed Citation: 11007482

Magie, C. R., et al. (1999). Mutations in the Rho1 small GTPase disrupt morphogenesis and segmentation during early Drosophila development Development 126: 5353-5364. PubMed Citation: 10556060

Nadarajan, S., et al. (2009). MSP and GLP-1/Notch signaling coordinately regulate actomyosin-dependent cytoplasmic streaming and oocyte growth in C. elegans. Development 136: 2223-2234. PubMed Citation: 19502484

Neer, E. J. (1995). Heterotrimeric G proteins: organizers of transmembrane signals. Cell 80: 249-257. PubMed Citation: 7834744

Offermanns S., et al. (1997). Impaired motor coordination and persistent multiple climbing fiber innervation of cerebellar purkinje cells in mice lacking galphaq. Proc. Natl. Acad. Sci. 94(25): 14089-14094. PubMed Citation: 9391157

Parks, S. and Wieschaus, E. (1991). The Drosophila gastrulation gene concertina encodes a G alpha-like protein. Cell 64: 447-58. PubMed Citation: 1899050

Pimplikar, S. W. and Simons, K. (1993), Regulation of apical transport in epithelial cells by the Gs class of heterotrimeric G protein. Nature 362: 456-458. PubMed Citation: 8385268

Quan, F., Wolfgang, W. J. and Forte, M. A. (1989). The Drosophila gene coding for the alpha subunit of a stimulatory G protein is preferentially expressed in the nervous system. Proc. Natl. Acad. Sci. 86: 4321-5. PubMed Citation: 2498884

Quan, F. and Forte, M. A. (1990). Two forms of Drosophila melanogaster Gs alpha are produced by alternate splicing involving an unusual splice site. Mol. Cell. Biol. 10: 910-7. PubMed Citation: 2106072

Quan, F., Thomas, L. and Forte, M. (1991). Drosophila stimulatory G protein alpha subunit activates mammalian adenylyl cyclase but interacts poorly with mammalian receptors: implications for receptor-G protein interaction. Proc. Natl. Acad. Sci. 88: 1898-902. PubMed Citation: 1848015

Quan, F, Wolfgang, W. J. and Forte, M. (1993). A Drosophila G-protein alpha subunit, Gf alpha, expressed in a spatially and temporally restricted pattern during Drosophila development. Proc. Natl. Acad. Sci. 90: 4236-40. PubMed Citation: 7683429

Renden, R. B. and Broadie, K. (2003). Mutation and activation of Galphas similarly alters pre- and postsynaptic mechanisms modulating neurotransmission. J. Neurophysiol. 89: 2620-2638. 12611964

Riedel, G., Casabona, G. and Reymann, K. G. (1995). Inhibition of long-term potentiation in the dentate gyrus of freely moving rats by the metabotropic glutamate receptor antagonist MCPG. J. Neurosci. 15: 87-98. PubMed Citation: 7823154

Scholich, K., et al. (1999). Facilitation of signal onset and termination by Adenylyl cyclase. Science 283(5406): 1328-1331. PubMed Citation: 10037603

Singh, K., Ju, J. Y., Walsh, M. B., DiIorio, M. A. and Hart, A. C. (2014). Deep conservation of genes required for both Drosophila melanogaster and Caenorhabditis elegans sleep includes a role for dopaminergic signaling. Sleep 37(9):1439-51 PubMed ID: 25142568

Sunahara, R. K., et al. (1997). Crystal structure of the adenylyl cyclase activator Gsalpha. Science 278(5345): 1943-1947. PubMed Citation: 9395396

Tolkacheva, T., et al. (1997). Cooperative transformation of NIH3T3 cells by G alpha12 and Rac1. Oncogene 15(6): 727-735. PubMed Citation: 9264413

Ueno, K., Kohatsu, S., Clay, C., Forte, M., Isono, K., Kidokoro, Y. (2006). Gsalpha is involved in sugar perception in Drosophila melanogaster. J. Neurosci. 26(23): 6143-52. 16763022

Wang, H.-Y., Watkins, D. C., and Malbon, C. C. (1992). Antisense oligodeoxynucleotides to Gs protein alpha-subunit sequence accelerate differentiation of fibroblasts to adipocytes. Nature 358: 334-337. 1379345

Warner, D. R., et al. (1999). Mutagenesis of the conserved residue Glu259 of Gsalpha demonstrates the importance of interactions between switches 2 and 3 for activation. J. Biol. Chem. 274(8): 4977-84. PubMed Citation: 9988742

Wenzel-Seifert. K., et al. (1998). Restricting mobility of Gsalpha relative to the beta2-adrenoceptor enhances adenylate cyclase activity by reducing Gsalpha GTPase activity. Biochem J. 334 ( Pt 3): 519-24. PubMed Citation: 9729456

van Meyel, D. J., Thomas, J. B. and Agulnick, A. D. (2003). Ssdp proteins bind to LIM-interacting co-factors and regulate the activity of LIM-homeodomain protein complexes in vivo. Development 130: 1915-1925. PubMed Citation: 12642495

Wolfgang, W. J., et al. (1990). Immunolocalization of G protein alpha-subunits in the Drosophila CNS. J. Neurosci. 10: 1014-24. PubMed Citation: 2108229

Wolfgang, W. J., et al. (1991). Restricted spatial and temporal expression of G-protein alpha subunits during Drosophila embryogenesis. Development 113: 527-38. PubMed Citation: 1782864

Wolfgang, W. J., et al (1996) Activation of protein kinase A-independent pathways by Gs-alpha in Drosophila. Proc. Natl. Acad. Sci. 93: 14542-47. 8962088

Wolfgang, W. J., et al. (2001). Genetic analysis of the Drosophila Gsalpha gene. Genetics 158: 1189-1201. 11454767

Wolfgang, W. J., Clay, C., Parker, J., Delgado, R., Labarca, P., Kidokoro, Y. and Forte, M. (2004). Signaling through Gs alpha is required for the growth and function of neuromuscular synapses in Drosophila. Dev Biol 268: 295-311. PubMed ID: 15063169

Zars, T., Wolf, R., Davis, R. and Heisenberg, M. (2000). Tissue-specific expression of a type I adenylyl cyclase rescues the rutabaga mutant memory defect: in search of the engram. Learn. Mem. 7: 18-31. 10706599

G protein salpha 60A: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 25 August 2012 

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