G protein α s subunit

Transcriptional regulation by CHIP/LDB complexes

It is increasingly clear that transcription factors play versatile roles in turning genes 'on' or 'off' depending on cellular context via the various transcription complexes they form. This poses a major challenge in unraveling combinatorial transcription complex codes. This study used the powerful genetics of Drosophila combined with microarray and bioinformatics analyses to tackle this challenge. The nuclear adaptor CHIP/LDB is a major developmental regulator capable of forming tissue-specific transcription complexes with various types of transcription factors and cofactors, making it a valuable model to study the intricacies of gene regulation. To date only few CHIP/LDB complexes target genes have been identified, and possible tissue-dependent crosstalk between these complexes has not been rigorously explored. SSDP proteins protect CHIP/LDB complexes from proteasome dependent degradation and are rate-limiting cofactors for these complexes. By using mutations in SSDP, 189 down-stream targets of CHIP/LDB were identified; these genes are enriched for the binding sites of Apterous (AP) and Pannier (PNR), two well studied transcription factors associated with CHIP/LDB complexes. Extensive genetic screens were performed and target genes were identified that genetically interact with components of CHIP/LDB complexes in directing the development of the wings (28 genes) and thoracic bristles (23 genes). Moreover, by in vivo RNAi silencing, novel roles were uncovered for two of the target genes, xbp1 and Gs-alpha, in early development of these structures. Taken together, these results suggest that loss of SSDP disrupts the normal balance between the CHIP-AP and the CHIP-PNR transcription complexes, resulting in down-regulation of CHIP-AP target genes and the concomitant up-regulation of CHIP-PNR target genes. Understanding the combinatorial nature of transcription complexes as presented here is crucial to the study of transcription regulation of gene batteries required for development (Bronstein, 2011).

Drosophila SSDP was identified on the basis of its ability to bind the nuclear adaptor protein CHIP/LDB (van Meyel, 2003; Chen, 2002). Both nuclear localization of SSDP and its ability to modulate the transcription activity of the CHIP-AP complex during wing development depend on its interaction with CHIP/LDB. This study implemented a combination of molecular, bioinformatic and genetic approaches that allowed has led to insight into the effect of SSDP on the transcriptional activity of CHIP/LDB complexes and their role in development. A genome wide screen was conducted for SSDP target genes in Drosophila using expression microarrays with mRNA isolated from larvae bearing hypomorphic alleles of ssdp. Analysis of transcription factor binding site enrichment served as an orthogonal assay that validates and extends the microarray results and thus contributes to understanding of the relation between the CHIP-AP and CHIP-PNR transcription complexes in specific tissues (e.g. wing and thorax) (Bronstein, 2011).

SSDP proteins directly bind DNA and mouse SSDP1 activates the expression of a reporter gene in both yeast and mammalian cells indicating that it is capable of regulating transcription activity. Enrichment was found for SSDP binding sites upstream of the genes identified in the microarray experiments on flies lacking SSDP. Moreover, in agreement with the positive transcriptional role of SSDP, enrichment for SSDP binding sites was restricted to the genes showing decreased expression in mutants. This strongly suggests that a significant number of these genes are bona fide SSDP target genes (Bronstein, 2011).

Consistent with the involvement of SSDP with the CHIP-AP complex, it was found that upstream regulatory regions of the SSDP putative target genes are also enriched for the AP binding site and the SSDP binding site. These sites are likely to be functionally significant, since loss of ssdp enhances the wing notching phenotype of a dominant allele of ap. Additionally, over-expression of Dlmo, whose product negatively regulates the CHIP-AP complex, also interacts with mutants of SSDP target genes, demonstrating that SSDP target genes are involved in the CHIP-AP pathway. The efficiency of finding genetic interactions among the genes differentially expressed in the microarray experiments, demonstrated the power of this approach. Specifically, 72% of the loci tested with Dlmo^Bx2 is more than an order of magnitude higher than an EP insertion screen (1.3% interacting) in a Dlmo^Bx1 sensitized background. Combined microarray and genetic loss of function screen allowed the identification of a similar number of Dlmo-interacting genes by screening a much smaller group of putative target genes (Bejarano, 2008). Of the 35 genes identified by Bejarano only CG1943 was found in the 189 genes identified in the current microarray screen. This study specifically identified down-stream targets of SSDP, while Bejarano searched for any modifiers of the Dlmo wing notching phenotype and thus uncovered genes that function in other regulatory pathways or genes that are upstream of the CHIP-AP complexes. This may explain the limited overlap between the current results and those of Bejarano (Bronstein, 2011).

In contrast to the enrichment of SSDP binding sites in the genes down-regulated in ssdp mutants, the PNR binding site was enriched specifically in the genes up-regulated in the ssdp mutants. A model is therefore presented in which loss of SSDP disrupts the balance between the CHIP-AP and CHIP-PNR complexes. Mammalian SSDP proteins protect LDB, LHX and LMO proteins from ubiquitination and subsequent proteasome-mediated degradation by interfering with the interaction between LDB and the E3 ubiquitin ligase, RLIM. It is therefore possible that in the absence of SSDP proteins, CHIP/LDB and LMO can escape degradation by interacting with GATA and beta-HLH proteins that are not subjected to proteasome-mediated regulation. The N-terminus of CHIP/LDB proteins is responsible for interaction with both PNR and RLIM. Thus, PNR/GATA proteins may partially interfere with the interaction between CHIP/LDB and RLIM making the CHIP/LDB-PNR/GATA complex more resistant to proteasome regulation and less dependant on the levels of SSDP proteins then the CHIP/LDB-LHX/AP complex (Bronstein, 2011).

According to the current model, in cells where both the CHIP-AP and CHIP-PNR complexes are active, loss of SSDP should result in the same phenotype as over-expression of PNR. Indeed, it was found that ssdp^L7/+ flies display duplications of scutellar sensory bristles, similar to gain of function mutations in pnr. In addition, lowered levels of pnr in ssdp^L7/+; pnr^VX6/+ flies suppresses scutellar bristle duplication. This indicates that the duplicated scutellar bristle phenotype of ssdp^L7/+ flies depends on the presence of PNR. As predicted by the model, since both AP and PNR regulate bristle formation, the functional interactions between SSDP target genes and ssdp^L7 and/or Chip^e5.5 resulted in either suppression or enhancement of the duplicated scutellar bristle phenotype (Bronstein, 2011).

These results in flies indicate that SSDP contributes differentially to CHIP/LDB complexes containing AP versus PNR. By contrast, mouse SSDP proteins positively contribute to the transcription activity and assembly of both LDB-GATA and LDB-LHX complexes, but the relative contribution of mammalian SSDP proteins to LDB complexes containing LHX proteins versus GATA proteins has not been specifically examined. It is possible that SSDP alters the balance of LIM-based CHIP/LDB complexes and GATA-containing CHIP/LDB complexes in the development of mice, as occurs in flies (Bronstein, 2011).

The search for enrichment of transcription factor binding sites upstream of the putative SSDP target genes identified additional transcription factors that may warrant future study. Some of these factors are associated with SSDP and CHIP/LDB complexes. For example, the binding sites for PNR and ZESTE (Z) were both enriched in the up-regulated putative SSDP target genes. This is in agreement with previous studies showing that Z can recruit the BRAHMA (BRM, the Drosophila homolog of the yeast SWI2/SNF2 gene) complex via its member OSA, which together negatively regulate the CHIP-PNR complex during sensory bristle formation through direct and simultaneous binding of OSA to both CHIP and PNR (Bronstein, 2011).

Some of the additional regulatory inputs at SSDP target genes may be evolutionarily conserved. For example, enrichment of STAT92E and SSDP binding sites was found in the down-regulated SSDP target genes. This may be significant, as a known role of ssdp is regulation of the JAK/STAT pathway during Drosophila eye development. Interestingly, mammalian STAT1 confers an anti-proliferative response to IFN-γ signaling by inhibition of c-myc expression. Similarly, expression of mammalian SSDP2 in human acute myelogenous leukemia cells and prostate cancer cells leads to cell cycle arrest and inhibits proliferation accompanied by down-regulation of C-MYC. These findings indicate that both in Drosophila and in mammals SSDP and STAT proteins have similar functions and may share common target genes (Bronstein, 2011).

While the transcription factor binding site analysis utilized all of the 189 putative SSDP target genes, genetic screens were conducted on a subset of them due to the availability of mutants. This suggests that more genetic interactions will be found among the untested genes. Even among this more limited subset, there are interesting new stories that suggest future experimental directions. For example, an insertion mutation in the Xbp1 gene suppressed the duplicated scutellar bristle phenotype characteristic of ssdp^L7/+ and Chip^e5.5/+ flies, indicating that XBP1 contributes positively to bristle formation. In contrast, when Xbp1 was silenced in ap-expressing cells both the wings and the scutum displayed a marked excess of sensory bristles while the scutellum was not affected. These results suggest that in the wing and scutum XBP1 acts as a negative regulator of bristle formation. Silencing of Xbp1 in pnr-expressing cells caused a similar excess of bristle on the scutum, accompanied by a reduced number of scutellar bristles, further emphasizing the opposing effects of XBP1 in these two distinct parts of the thorax. Such contrasting phenotypes have been previously documented for several pnr mutants as well. In flies and mammals XBP1 regulates the ER stress response, also termed the unfolded protein response (UPR). Since one of the functions of the ER is the production of secreted proteins, UPR-related pathways are widely utilized during the normal differentiation of many specialized secretory cells. In this respect it would be interesting to examine whether SSDP and CHIP/LDB complexes affect the production of secreted morphogens, such as Wingless (WG), the secreted ligands of the EGFR receptor, Spitz (SPI) and Argos (AOS), or the secreted Notch binding protein Scabrous (SCA) via XBP1 during wing and sensory bristle formation. Alternatively, the transcription factor XBP1 may directly regulate the expression of genes required for differentiation of the wing and sensory bristles. Indeed, carbohydrate ingestion induces XBP1 in the liver of mice, which in turn directly regulates the expression of genes involved in fatty acid synthesis. This role of XBP1 is independent of UPR activation and is not due to altered protein secretory function. Curiously, the two GO function categories 'cellular carbohydrate metabolism' and 'cellular lipid metabolism' which are enriched among Xbp1 target genes in mouse skeletal muscle and secretory cells were also enriched in the list of putative SSDP target genes. Whether this reflects a secondary effect due to the down-regulation of Xbp1 in ssdp mutants or a direct regulation of these processes by SSDP is yet to be determined (Bronstein, 2011).

Additional novel functions for CHIP/LDB complexes are implied by the results regarding the Gs-alpha60A (a.k.a. CG2835) gene. G protein coupled receptors are important regulators of development by for example, signaling via the protein kinase A (PKA) pathway. Activation or inhibition of PKA signaling during pupal wing maturation perturb proper adhesion of dorso-ventral wing surfaces resulting in wing blistering. This phenotype may be due to miss-regulation of wing epithelial cell death in ap-expressing cells. Interestingly, similar wing blisters occur in the wing of Dlmo^Bx2 flies. Moreover, it was found that mutant alleles of Gs-alpha60A enhanced the wing blistering phenotype of Dlmo^Bx2. Silencing of G-salpha60A in ap-expressing cells caused a curled wing phenotype. Such a phenotype can result from differences in the size of the dorsal and ventral wing blade surfaces. In addition, silencing of this gene in pnr-expressing cells caused the posterior pair of scutellar bristles to form in reversed orientation. Bristle orientation have been proposed to be regulated by planar cell polarity genes. Taken together these results point to novel aspects of regulation of wing and sensory bristle development by SSDP and CHIP/LDB complexes mediated by G-alpha proteins (Bronstein, 2011).

This genome-wide expression profiling and bioinformatics analysis of ssdp mutant larvae, combined with genetic screens resulted in gained insight into the intricate context-dependent transcriptional regulation by CHIP/LDB complexes. It was possible to identify 28 putative SSDP target genes that are involved in wing development and 23 putative SSDP target genes that play a role in scutellar bristle formation. Examination of two of these, xbp1 and Gs-alpha60A, suggests novel aspects of developmental regulation such as the involvement of SSDP and CHIP/LDB complexes in ER function and PKA signaling. Furthermore, it was shown that SSDP proteins contribute differentially to transcription activity, and probably to the balance in formation of CHIP-AP and CHIP-PNR complexes. Furthermore potential novel partners of SSDP in regulating transcription of downstream genes during fly development were. It stands to reason that an extension of the genetic analysis to mammals and other vertebrates will reveal a host of additional functions of SSDP and CHIP/LDB during the multifaceted process of transcriptional regulation that underlies the development of multicellular organisms (Bronstein, 2011).

G(o) signaling is required for Drosophila associative learning

Heterotrimeric G(o) is one of the most abundant proteins in the brain, yet relatively little is known of its neural functions in vivo. This study demonstrates that G(o) signaling is required for the formation of associative memory. In Drosophila, pertussis toxin (PTX) is a selective inhibitor of G(o) signaling. The postdevelopmental expression of PTX within mushroom body neurons robustly and reversibly inhibits associative learning. The effect of G(o) inhibition is distributed in both γ- and α/β-lobe mushroom body neurons. However, the expression of PTX in neurons adjacent to the mushroom bodies does not affect memory. PTX expression also does not interact genetically with a rutabaga adenylyl cyclase loss-of-function mutation. Thus, G(o) defines a new signaling pathway required in mushroom body neurons for the formation of associative memory (Ferris, 2006).

An associative memory is one that links external stimuli to particular events, such that the stimuli come to predict the events. In the negatively reinforced olfactory associative learning assay of Drosophila, flies are presented with an odor (conditional stimulus paired, CS⁺) paired with an electric shock (unconditioned stimulus, US). The flies are then presented with a second odor (conditioned stimulus unpaired, CS^-). The associative memory is measured as the conditioned avoidance of the CS⁺ in a T-maze. The disruption of the cyclic AMP (cAMP) signaling pathway within Drosophila leads to reduced learning scores. The effect of cAMP disruption has been mapped back to the mushroom body neurons through the targeted expression of a constitutively active G(s)α and by rescuing the rutabaga type I adenylyl cyclase (rut) phenotype with targeted expression of a rut cDNA. It is thought that the cAMP pathway controls the association between the CS⁺ and the US within the mushroom body neurons (Ferris, 2006).

The G(o) heterotrimeric protein is thought to be the most abundant membrane protein in the vertebrate brain and is activated both by numerous G protein-coupled receptors (GPCRs) and by amyloid precursor protein. Although G(o) can participate in diverse signaling pathways, only a few specific in vivo functions have been ascribed to this molecule. In Drosophila, G(o)α47A is the only gene encoding the alpha subunit of G(o), and it is expressed throughout the adult brain. The G(o) protein is much more abundant in the heads of rutabaga (rut) and dunce learning mutants than in the heads of wild-type flies, suggesting a possible role for G(o) in memory formation (Ferris, 2006).

The S1 subunit of PTX from Bordetella pertussis catalyzes the transfer of an ADP-ribose onto the Gα subunit of the vertebrate G(i/o/t) heterotrimeric G proteins, preventing these proteins from binding to activated GPCRs. In Drosophila, PTX is a selective enzymatic inhibitor of G(o) signaling: Drosophila does not have a transducin homolog, and the G(i)α65A protein does not contain the PTX recognition site, whereas G(o)α does; PTX will ADP-ribosylate a single protein in Drosophila, as seen in western blots and after isoelectric focusing; and PTX comigrates with G(o)α and is immunoprecipitated by independent G(o)α-specific antibodies (Ferris, 2006).

To determine if G(o) is a mediator of associative memory, a PtxA transgene was expressed within the mushroom body neurons. The P{UASPTX}16 transgenic line was selected because the basal expression of PTX is low in this line and because PTX can be induced by Gal4, albeit in small amounts. G(o)α47A loss-of-function mutant embryos die during embryogenesis owing to defects in nervous system and mesoderm development. In keeping with this result, it was found that the induction of PTX within the developing mesoderm or nervous system also results in embryonic lethality, indicating that this toxin is functional when expressed early in development (Ferris, 2006).

The role of G(o) in associative memory was examined by inducing PTX expression within the adult mushroom bodies with the P{MBSwitch}12 Gene-Switch driver. The resulting induction abolished the immediate associative memory 3 min after training, which is frequently taken as a measure of learning. Although the PTX-uninduced P{MBSwitch}12/P{UASPTX}16 flies also showed reduced learning, their scores were not significantly lower than the PTX-uninduced P{UASPTX}16/+ control group. The induction of PTX within the mushroom body did not alter naïve sensitivities to either odorants or electric shock, indicating that PTX expressed in the mushroom bodies does not affect the perception of the stimuli (Ferris, 2006).

The severity of the PTX learning phenotype might result from the death of the mushroom body neurons. This hypothesis was tested by examining the integrity of the mushroom bodies after the induction of PTX and by establishing whether the associative learning phenotype was reversible. Because Gene-Switch has slow off-rate kinetics, the Gal80^ts system was used with the P247 Gal4 driver. P247 drives expression in ~700 α/β- and γ-lobe mushroom body neurons. Two independent Gal80^ts transgenes were used to ensure more complete inhibition of Gal4 at 18°C. After inducing PTX for 12 h at 32°C, 3-min memory was almost entirely abolished. Using antibodies to downstream of receptor kinase (DRK), which preferentially mark the mushroom body, it was found that PTX induction did not alter either the gross structure of the mushroom bodies or the expression of DRK. Similar results were found using antibodies to cAMP-dependent protein kinase 1 (DCO). It was also found that the effect of PTX was reversible: although a 2-h induction of PTX within mushroom body neurons produced significant inhibition of 3-min memory, this effect was completely reversed after 6 d. Therefore, the effect of PTX on learning is not due to the death of the mushroom body neurons (Ferris, 2006).

Next, whether the effect of PTX on learning was specific to the mushroom bodies was examined. PTX was induced in the R3 and R4d neurons of the ellipsoid body and separately in the dorsally paired medial (DPM) neurons, which innervate the mushroom bodies. The induction of PTX with the Gal80^ts system in either set of neurons did not affect performance in the learning assay, suggesting that PTX is cell autonomous. Moreover, PTX induction in the DPM neurons had no effect on 60-min memory. The inhibition of neurotransmission in DPM neurons by the shibire^ts transgene completely blocks 60-min memory but has no effect on 3-min memory. Thus, PTX and shibire^ts have different effects in the DPM neurons, indicating that PTX is not a general inhibitor of neurotransmission (Ferris, 2006).

Experiments were conducted to see whether the requirement for G(o) signaling in olfactory associative learning is dispersed throughout the different neurons of the mushroom body lobes, or if the requirement is limited to a subset of these neurons. Several genes have been identified that are preferentially expressed in the different mushroom body lobes, indicating that these lobes have distinct molecular repertoires; however, direct tests for lobe function have yet to provide unequivocal and differentiated roles for the constituent neurons in associative learning. The c772 Gal4 line drives expression in ~800 neurons of the α/β and γ lobes. It was found that a 12-h induction with c772 was sufficient to ablate the associative memory, whereas a 2-h induction was not. There were no differences in the naïve avoidance of odor or shock between the c772/Gal80^ts20; PTX/Gal80^ts2 PTX-induced and PTX-uninduced experimental groups. There were, however, some differences in naïve odor avoidance between the c772/Gal80^ts20; PTX/Gal80^ts2 PTX-induced group and the control genotypes, suggesting that PTX induction in non-mushroom-body neurons by c772 may affect odor perception or discrimination and that the Gal80^ts inhibition may not be complete in these neurons. The differences in odor avoidance may also participate in the severe c772/PTX phenotype, although it is unlikely to have a major effect on learning as naïve avoidance scores were not significantly different in the within-genotype control group. It is likely that differences in expression levels between c772 and P247 account for the different time courses in the inhibition of learning by PTX between these two lines. The 12-h induction of PTX in the γ-lobe neurons marked by 1471 caused a substantial, but not complete, loss of 3-min memory, as did the expression of PTX in the α/β-lobe neurons marked by c739. Thus, G(o) signaling is required for 3-min memory in both the γ and α/β neurons of the mushroom body as defined by the 1471 and c739 drivers, respectively. In contrast, PTX driven by the α/β-lobe driver 17d did not have an observable effect on 3-min memory. The mushroom body neurons defined by 17d are most likely the core neurons of the α/β lobe, which may be functionally distinct from the other neurons of the α/β lobe, since they are insensitive to the effects of PTX in associative memory and have no effect on the rescue of the rut learning phenotype. The fact that associative memory formation was affected by PTX induction in the α/β- and γ-lobe neurons, but not in the putative α/β core neurons, defines a new requirement for G(o) signaling in these lobes for learning and memory and should further help dissect the memory process in these neurons (Ferris, 2006).

Next it was considered whether the G(o) pathway interacts genetically with the rut adenylyl cyclase in associative memory formation. The persistent activation of vertebrate G(o) may initially lead to the short-term inhibition of type I adenylyl cyclase, followed by the increased responsiveness of this enzyme to G(s)α stimulation, known as heterologous sensitization or supersensitization. Thus, PTX may be interfering with the down regulation of rut activity by G(o), resulting in neurons with too much adenylyl cyclase activity. Alternatively, PTX may inhibit the heterologous sensitization of rut by G(o), leaving the neurons with too little cAMP after G(s) activation. The former hypothesis predicts that a reduction in rut activity may partially suppress the PTX phenotype, whereas the latter suggests that the reduction in rut may act synergistically with PTX. These predictions were tested by looking for a genetic interaction between a mild induction of PTX in the subset of mushroom body neurons defined by P247 and a single copy of rut²⁰⁸⁰. This rut mutation demonstrates a semidominant haploinsufficiency, indicating that learning is extremely sensitive to the activity levels of this enzyme. It was found that the performance of the rut²⁰⁸⁰/+; Gal80^ts20/+; PTX/P247, Gal80^ts2 PTX-induced flies was reduced, but not significantly, as compared to that of the Gal80^ts20/+; PTX/P247, Gal80^ts2 flies. This result suggests an additive interaction between PTX and the rut²⁰⁸⁰ heterozygote, but there was evidently neither suppression nor a synergistic relationship between PTX and one copy of rut²⁰⁸⁰. The independence of G(o) function during learning from rut was further assessed in rut homozygotes. The performance of the rut²⁰⁸⁰; Gal80^ts20/+; PTX/P247, Gal80^ts2 PTX-induced flies was significantly worse than that of either the PTX-induced flies or the rut homozygous flies. Thus, G(o) signaling has functions in olfactory learning and memory within the mushroom body neurons defined by P247 that are independent of rut (Ferris, 2006).

This study has shown, through the postdevelopmental induction of PTX expression within mushroom bodies, that activation of G(o) is required during the physiological events, which lead to associative memory formation. The severity of the learning phenotype in PTX-induced flies coupled with the lack of genetic interaction with rut²⁰⁸⁰ strongly suggests that the function of G(o) in associative learning and memory is largely independent of the cAMP pathway. Additional members of this new associative learning pathway are currently unknown. One possibility is that, similar to the role of the G(o) in the vertebrate dorsal root ganglia, the Drosophila G(o) may participate in learning through the inhibition of voltage-gated Ca²⁺ channels (VGCCs). These Ca²⁺ channels are thought to be activated by the odor-induced depolarization of the mushroom body neurons, leading to the release of synaptic vesicles and the CS pathway activation of rut. It is plausible that the negative regulation of the VGCCs may be necessary to restrict the number of activated synapses during learning. Nevertheless, it is now clear that the in vivo functions of G(o) include the formation of associative memories in Drosophila (Ferris, 2006).

Gsalpha is involved in sugar perception in Drosophila melanogaster

In Drosophila, gustatory receptor genes (Grs) encode G-protein-coupled receptors (GPCRs) in gustatory receptor neurons (GRNs) and some olfactory receptor neurons. One of the Gr genes, Gr5a, encodes a sugar receptor that is expressed in a subset of GRNs and has been most extensively studied both molecularly and physiologically, but the G-protein alpha subunit (Galpha) that is coupled to this sugar receptor remains unknown. This study proposes that Gs is the Galpha that is responsible for Gr5a-mediated sugar-taste transduction, based on the following findings: (1) immunoreactivities against Gs were detected in a subset of GRNs including all Gr5a-expressing neurons. (2) trehalose-intake is reduced in flies heterozygous for null mutations in DGsalpha, a homolog of mammalian Gs, and trehalose-induced electrical activities in sugar-sensitive GRNs were depressed in those flies. Furthermore, expression of wild-type DGsalpha in sugar-sensitive GRNs in heterozygotic DGsalpha mutant flies rescues those impairments. (3) Expression of double-stranded RNA for DGsalpha in sugar-sensitive GRNs depresses both behavioral and electrophysiological responses to trehalose. Together, these findings indicate that DGsalpha is involved in trehalose perception. It is suggested that sugar-taste signals are processed through the Gsalpha-mediating signal transduction pathway in sugar-sensitive GRNs in Drosophila (Ueno, 2006: full text of article).

It was found that DGsalpha is localized not only in Gr5a-GRNs but also in non-Gr5a GRNs (~40 GRNs in a labelum). In labela, there are at least four types of GRNs sensitive to sugar, low concentrations of salt, bitter-substances/high concentrations of salt, water, and mechanosensory neurons. Then, two questions arise: (1) which GRN, other than Gr5a-GRNs, contains DGsalpha? (2) Is DGsalpha in unknown GRNs involved in the taste signaling of GRNs? The behavioral responses to bitter solutions were not different between heterozygous DGsalpha-null mutant and control flies, and the behavioral and electrophysiological responses to water were not different among all DGsalpha strains examined in this study. It is known that salt responses in larvae require amiloride-sensitive channels encoded by ppk11 and ppk19, and the low and high concentrations of salt responses do not require Ggamma1 in adult flies. These findings together with the current results suggest that DGsalpha in non-Gr5a GRNs serves for other signaling than taste or that the non-Gr5a GRNs containing DGsalpha are mechanosensory neurons. However, because the bitter and water responses in the homozygous DGsalpha^R19 mutant were not studied, the possibility that DGsalpha is involved in bitter and/or water tastes cannot be rigorously excluded (Ueno, 2006).

It is suggested that, in Drosophila, the Gs-mediated cAMP transduction pathway is the main signaling route in sugar-sensitive GRNs. In contrast, the PLC/IP₃ mediating pathway is involved in sugar-taste signaling in the fleshfly (Boettcherisca peregrina) and the guanosine-3',5'-cyclic monophosphate/nitric oxide pathway in the blowfly (Phormia regina). The cAMP pathway may be involved in sugar-taste perception in the frog, rat, and pig, whereas a recent study on T1R2/T1R3 gustatory sugar receptors of the mouse supports involvement of the PLC pathway. Additional comparative studies are necessary to elucidate the diversity of molecular mechanisms of sugar-taste signaling in various animals (Ueno, 2006).

Protein Interactions

The long and short forms of Drosophila G salpha subunits were expressed in murine cultured cells lacking endogeneous Gs alpha subunits. Both subunits stimulate mammalian adenylyl cyclase (Drosophila homolog: Rutabaga). A constitutively active G salpha, mutated in the putative S box of the guanine nucleotide-binding domain was tested for activation of adenylyl cylcase. Relative to wild type G salpha, basal activity was increased 12-fold. Constitutively active G salpha therefore results in the constitutive activation of mammalian adenylyl cyclase. Activated G salpha subunits have been shown to potentiate the ability of forskolin to stimulate the activity of adenylyl cyclase. This effect is also observed in cultured cells expressing the fly and rat G salpha subunits. G salpha subunits are susceptible to modification by cholera toxin at an internal arginine residue, resulting in a reduction in the intrinsic GTPase activity of the alpha subunit and constitutive activity. The conservation of the susceptible arginine of the fly G salpha subunits and the high homology of the invertebrate and vertebrate proteins in the region flanking this residue suggests that the fly's G salpha subunits may also be cholera toxin substrates. The Drosophila G salpha subunits, like their mammalian counterparts, do turn out to be substrates for functional modification by cholera toxin. Activation of transduced cells with isoproterenol, a ß-adrenergic agonist results in a small increase in adenylyl cyclase activity; this activation is stimulated by both long and short Drosophila Gs alpha isoforms. The prostaglandin E1 receptor fails to stimulate adenylyl cyclase through Drosophial Gs alpha. The small increase with the ß-adrenergic agonist, and the lack of effect of prostaglandin E1 indicates that the interaction of Drosophila Gs alpha subunits with mammalian G-protein-coupled receptors is generally inefficient (Quan, 1991).

A neuropeptide signaling pathway regulates synaptic growth in Drosophila

Neuropeptide signaling is integral to many aspects of neural communication, particularly modulation of membrane excitability and synaptic transmission. However, neuropeptides have not been clearly implicated in synaptic growth and development. This study demonstrates that cholecystokinin-like receptor (CCK-like receptor at 17D1; CCKLR), and Drosulfakinin (DSK), its predicted ligand, are strong positive growth regulators of the Drosophila melanogaster larval neuromuscular junction (NMJ). Mutations of CCKLR (CCKLR-17D1 but not CCKLR-17D3) or dsk produce severe NMJ undergrowth, whereas overexpression of CCKLR causes overgrowth. Presynaptic expression of CCKLR is necessary and sufficient for regulating NMJ growth. CCKLR and dsk mutants also reduce synaptic function in parallel with decreased NMJ size. Analysis of double mutants revealed that DSK/CCKLR regulation of NMJ growth occurs through the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA)-cAMP response element binding protein (CREB) pathway. These results demonstrate a novel role for neuropeptide signaling in synaptic development. Moreover, because the cAMP-PKA-CREB pathway is required for structural synaptic plasticity in learning and memory, DSK/CCKLR signaling may also contribute to these mechanisms (Chen, 2012).

Proper synaptic growth is essential for normal development of the nervous system and its function in mediating complex behaviors such as learning and memory. The Drosophila larval neuromuscular junction (NMJ) has become a powerful system for studying the molecular mechanisms underlying synaptic development and plasticity, and many of the key synaptic proteins are evolutionarily conserved (Chen, 2012).

Genetic and molecular analysis in Drosophila has uncovered numerous molecules and pathways that regulate NMJ growth, including proteins required for cell adhesion, endocytosis, cytoskeletal organization, and signal transduction via TGF-β, Wingless, JNK, cAMP, and other signaling molecules. For example, previous studies revealed that increased cAMP levels led to a down-regulation of the cell adhesion protein FasII at synapses, and the activation of the cAMP response element binding protein (CREB) transcription factor to achieve long-lasting changes in synaptic structure and function. Despite the identification and characterization of these various positive and negative regulators, understanding of the networks that govern synaptic growth is still incomplete, with many of the key components and mechanisms yet to be uncovered and analyzed (Chen, 2012).

To search for new regulators of synaptic growth, a forward genetic screen was conducted for mutants exhibiting altered NMJ morphology. In this screen, a new mutation was discovered that exhibits strikingly undergrown NMJs, which indicates disruption of a positive regulator of NMJ growth. The affected protein was identified as cholecystokinin (CCK)-like receptor (CCKLR), a putative neuropeptide receptor that belongs to the family of G-protein coupled receptors (GPCRs) sharing a uniform topology with seven transmembrane domains. When activated by their ligands, neuropeptide GPCRs affect levels of second messengers such as cAMP, diacylglycerol, inositol trisphosphate, and intracellular calcium. Through activation of their cognate receptors, secreted neuropeptides mediate communication among various sets of neurons as well as other cell types to regulate several physiological activities, including feeding and growth, molting, cuticle tanning, circadian rhythm, sleep, and learning and memory. In general, neuropeptides act by modulating neuronal activity through both short-term and long-term effects. Short-term effects include modifications of ion channel activity and alterations in release of or response to neurotransmitters. Long-term effects include changes in gene expression through activation of transcription factors and protein synthesis. In contrast with the well-known effects of neuropeptide signaling on neuronal activity and the strength of synaptic transmission, regulation of synaptic growth and development by neuropeptides has not previously been clearly established (Chen, 2012).

This study demonstrates that CCKLR is required presynaptically to promote NMJ growth. Moreover, mutations of drosulfakinin (dsk), which encode the predicted ligand of CCKLR, cause similar NMJ undergrowth and interact genetically with CCKLR mutations, indicating that DSK and CCKLR are components of a common signaling mechanism that regulates NMJ growth. In addition to the morphological phenotypes caused by mutations of CCKLR and dsk, mutant larvae also exhibit deficits in synaptic function. Through phenotypic analysis of double mutant combinations, it was shown that DSK/CCKLR signals through the cAMP-PKA-CREB pathway to regulate NMJ growth. The results suggest a novel role for neuropeptide signaling in regulation of synaptic development. Moreover, because the cAMP-PKA-CREB pathway is required for structural plasticity of synapses in learning and memory, DSK/CCKLR signaling may also contribute to these mechanisms (Chen, 2012).

Neuropeptides, whose effects have been extensively studied at NMJs, are usually described as neuromodulators because they modify the strength of synaptic transmission. For example, proctolin can potentiate the action of glutamate at certain NMJs in insects. However, involvement of neuropeptides in regulating neural development has not been well characterized. Recently, a C-type natriuretic peptide acting through a cGMP signaling cascade was found to be required for sensory axon bifurcation in mice, which suggests that neuropeptides may have a broader role in development than previously appreciated. The current studies demonstrate that DSK and its receptor, CCKLR, are strong positive regulators of NMJ growth in Drosophila (Chen, 2012).

DSK belongs to the family of FMRFamide-related peptides (FaRPs), which is very broadly distributed across invertebrate and vertebrate phyla. Originally identified in clams, FaRPs affect heart rate, blood pressure, gut motility, feeding behavior, and reproduction in invertebrates. They have been shown to enhance synaptic efficacy at NMJs in locust and to modulate presynaptic Ca²⁺ channel activity in crustaceans. In Drosophila, various neuropeptides derived from the FMRFa gene can modulate the strength of muscle contraction when perfused onto standard larval nerve-muscle preparations. To these previously described functions of FaRPs, this study adds a new role as a positive regulator of NMJ development (Chen, 2012).

Transgenic rescue experiments, RNAi expression, and overexpression of WT CCKLR demonstrate that CCKLR functions presynaptically in motor neurons to promote NMJ growth. Downstream components of this pathway were identified on the basis of known biochemistry of GPCRs and phenotypic interactions in double mutant combinations. GPCRs typically function by activating second messenger pathways via G proteins. Because loss-of-function mutations in dgs (which encodes the Gsα subunit in Drosophila) cause NMJ undergrowth, it is hypothesized that CCKLR signals through Gsα. Consistent with this idea, it was found that presynaptic constitutively active dgs overexpression rescues the NMJ undergrowth phenotype of CCKLR mutants. Conversely, dominant dose-dependent interactions were observed between CCKLR-null mutations and mutations of rut, which encodes an AC; or PKA-C1, which encodes a cAMP-dependent protein kinase, resulting in significant reductions in NMJ growth. These data place CCKLR together with the other genes in a common cAMP-dependent signaling pathway that regulates NMJ growth (Chen, 2012).

It is known that the AC encoded by rut is activated by Gsα, and on the basis of the results, it is proposed that Gsα is downstream of CCKLR signaling. However, the NMJ undergrowth in rut¹, which is a presumptive null mutant, is not as severe as that of a CCKLR-null mutant. This is likely due to the fact that the Drosophila genome contains up to seven different AC-encoding genes, all of which are stimulated by Gsα. Presumably, one or more additional AC-encoding genes share some functional overlap with rut in regulation of NMJ growth. This idea is in good agreement with the results of Wolfgang (2004), who found that the NMJ undergrowth phenotype of rut¹ is weaker than that of dgs mutants, which they also interpreted as an indication that multiple ACs are activated by the Gsα encoded by dgs (Chen, 2012).

The primary effector of this pathway is CREB2, a transcriptional regulatory protein that is activated upon phosphorylation by PKA. Consistent with the idea that CCKLR ultimately acts via activation of CREB, loss-of-function mutations of dCreb2 or neuronal overexpression of a dominant-negative dCreb2 transgene cause NMJ undergrowth similar to that of CCKLR-null mutants. Additionally, loss of one copy of dCreb2 in a CCKLR heterozygous background also causes NMJ undergrowth, and overexpression of WT dCreb2 fully rescues the NMJ undergrowth phenotype of CCKLR null, even leading to NMJ overgrowth. Thus, regulation of NMJ growth through the CCKLR signaling pathway is clearly mediated by dCreb2, whose activity is itself necessary and sufficient for regulating NMJ growth. This conclusion differs from another study that suggested that dCreb2 is required for NMJ function but not NMJ growth. One possible explanation for this discrepancy is that a weaker, inducible heat shock-driven transgene was used to express dCreb2 in the earlier work, whereas strong constitutive neuronal drivers were used this study. In any case, the current results demonstrate that in addition to its known role in NMJ function, CREB2 is also a strong positive regulator of NMJ growth and is likely to play a greater role in structural plasticity of synapses in learning and memory in Drosophila than previously suggested. This conclusion is consistent with a recent study indicating that sprouting of type II larval NMJs in response to starvation is stimulated by a cAMP/CREB-dependent pathway via activation of an octopamine GPCR (Chen, 2012).

In addition to being undergrown, NMJs in CCKLR mutant larvae also exhibit a functional deficit. This is perhaps less straightforward than it might seem. Previous analyses of mutations affecting growth of the larval NMJ in Drosophila have shown that there is no simple correlation between the size and complexity of the NMJ and the amplitude of EJPs or amount of neurotransmitter release. These discrepancies arise because of various homeostatic compensatory mechanisms and because some of the affected signaling pathways alter synaptic growth and synaptic function in different ways via distinct downstream targets. For example, a mutation in highwire, which has the most extreme NMJ overgrowth phenotype described, is associated with a decrease in synaptic transmission. Wallenda mutations have been shown to fully suppress the overgrowth phenotype, but have no effect on the deficit in synaptic transmission (Chen, 2012).

In the case of CCKLR mutants, however, there appears to be a very good correspondence between the morphological phenotype and the electrophysiological phenotype: the reduction in the total number of active zones in CCKLR larvae as measured morphologically correlates very well with the reduction in quantal content that was observe. In addition, no difference in CCKLR mutants was detected in calcium sensitivity of transmitter release or in the size or frequency of spontaneous release events. Thus, the synaptic growth phenotype of CCKLR mutant NMJs is sufficient to account for the functional phenotype. However, the possibility cannot be ruled out that DSK/CCKLR signaling also exerts some modulatory effect on NMJ function that is distinct from its effect on NMJ development (Chen, 2012).

DSK is identified as the Drosophila orthologue of CCK, the ligand of CCKLR in vertebrates, on the basis of sequence analysis. The genetic analysis strongly supports the conclusion that DSK is the ligand of CCKLR at the larval NMJ. First, mutations of dsk and expression of dsk RNAi result in NMJ undergrowth phenotypes similar to that of CCKLR mutants. Second, loss of one copy of both dsk and CCKLR in double heterozygotes results in NMJ undergrowth. Third, heterozygosity for dsk does not further enhance the phenotype of a CCKLR-null mutant as expected if DSK regulates NMJ growth through its action on CCKLR. Fourth, overexpression of UAS-dsk does not rescue the undergrowth phenotype of a CCKLR-null mutant, but CCKLR overexpression can rescue NMJ undergrowth of a dsk hypomorphic mutant (Chen, 2012).

The discovery of an entirely novel role for neuropeptide signaling in NMJ growth raises several questions about how this signaling is regulated and the biological significance of this mechanism. Although answers to these questions will require much additional work, an immediate question is whether a paracrine or autocrine mechanism is involved. In the case of octopamine-mediated synaptic sprouting in response to starvation, both autocrine and paracrine signaling are involved in the sprouting of type II and type I NMJs, respectively. In an early immunohistochemical investigation, it was reported that DSK was detected in medial neurosecretory cells in the larval CNS that extended projections anteriorly into the brain and posteriorly to the ventral ganglion. As it was not possible to obtain the original DSK antiserum and raising a new antiserum was not successful, the previous report has not been extended or confirmed. Instead, tissue-specific RNAi experiments were performed to examine the spatial requirement for DSK. Pan-neuronal dsk RNAi expression indicates that DSK expression in neurons is required to promote NMJ growth. In addition, C739-Gal4-driven dsk RNAi also causes NMJ undergrowth, whereas OK-Gal4-driven dsk RNAi in motor neurons does not. The expression pattern of C739-Gal4 overlaps with the DSK-positive cells previously identified by immunohistochemistry, which suggests that DSK produced by those neurosecretory cells is required for normal NMJ growth. Thus, from available data, it seems most likely that DSK is acting in paracrine fashion to regulate NMJ growth. However, further investigation will be necessary to determine the exact source of the DSK that promotes NMJ growth to fully understand how this neuropeptide regulates NMJ development (Chen, 2012).

Studies have demonstrated a role for CREB in long-term synaptic plasticity—structural changes in synaptic morphology that underlie the formation of long-term memories. This study shows that in addition to CREB's role in structural modification of synapses in response to experience after development is complete, it is also a key regulator of growth and morphology during development of the larval NMJ. Moreover, although CREB is the transcriptional effector for many GPCRs, the fact that NMJs in CCKLR mutants are as undergrown as those of CREB mutants suggests that DSK/CCKLR signaling is a major input to CREB during NMJ growth. Many of the genes encoding intermediate components of the pathway such as dnc, rut, and PKA also have effects on NMJ growth and development as well as on synaptic plasticity and learning and memory, further emphasizing an overlap between the mechanisms that regulate synaptic growth during development and those that regulate postdevelopmental structural synaptic plasticity. These results raise the possibility that DSK/CCKLR signaling also plays a role in long-term synaptic plasticity and learning as well as in synaptic development (Chen, 2012).

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