InteractiveFly: GeneBrief

inactivation no afterpotential E: Biological Overview | References

BIOLOGICAL OVERVIEW

In Drosophila, a phospholipase C-mediated signaling cascade links photoexcitation of rhodopsin to the opening of the TRP/TRPL channels. A lipid product of the cascade, diacylglycerol (DAG) and its metabolite(s), polyunsaturated fatty acids (PUFAs), have both been proposed as potential excitatory messengers. A crucial enzyme in the understanding of this process is likely to be DAG lipase (DAGL). However, DAGLs that might fulfill this role have not been previously identified in any organism. In this work, the Drosophila DAGL gene, inaE, has been identified from mutants that are defective in photoreceptor responses to light. The inaE-encoded protein isoforms show high sequence similarity to known mammalian DAG lipases, exhibit DAG lipase activity in vitro, and are highly expressed in photoreceptors. Analyses of norpA inaE double mutants and severe inaE mutants show that normal DAGL activity is required for the generation of physiologically meaningful photoreceptor responses (Leung, 2008).

Visual transduction in Drosophila utilizes a G protein-coupled, phospholipase C-mediated signaling cascade. Phospholipase C, upon activation via rhodopsin and G protein, Gq, catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) into two potential second messengers, diacylglycerol (DAG) and inositol trisphosphate (IP₃). A body of evidence suggests that IP₃ is not involved in Drosophila phototransduction, leaving the DAG branch as a likely source of messenger(s) of activation for the phototransduction channels, transient receptor potential (TRP) and TRP-like (TRPL). The mechanism by which the diacylglycerol (DAG) branch might activate the TRP/TRPL channels is still unresolved. The first indication that a lipid messenger might be involved was provided by Chyb (1999), who showed that polyunsaturated fatty acids (PUFAs) could activate both TRP and TRPL channels either in intact photoreceptors or heterologous expression systems. Later, evidence was provided that DAG is required for photoreceptor excitation using DAG kinase mutants, rdgA. Because the conversion of DAG to phosphatidic acid is blocked in these mutants, they should have an elevated DAG basal level. TRP/TRPL channels are constitutively active in rdgA (Raghu, 2000), and diminished responses of hypomorphic PLC (norpA) mutants can be greatly enhanced by rdgA mutations (Hardie, 2002), in support of the contention that DAG might be excitatory to the channels. However, rdgA mutations are expected to raise the basal levels of not only DAG but also its metabolites. In addition to these two molecules, phosphatidylinositol 4,5-bisphosphate (PIP₂) has also been suggested to play a role in channel excitation (for review see Hardie, 2003). Currently, no consensus exists as to which, if any, of these might be the excitatory agent for TRP/TRPL channels (Leung, 2008).

Drosophila TRP is the founding member of a superfamily of TRP channel proteins. There are now nearly 30 mammalian members of this superfamily comprising seven subfamilies. Although these channels are heterogeneous in their modes of activation, at least four mammalian TRP channels have been reported to be activated by DAG: TRPC2, -3, -6, and -7. While there may be variations in the mechanisms of activation of these channels, elucidation of Drosophila TRP/TRPL channel activation could provide insight into activation of these channels as well (Leung, 2008).

Because both DAG and its potential metabolite, PUFA, have been implicated in the activation of TRP/TRPL channels, a key enzyme in this process is likely to be DAG lipase, which catalyzes the hydrolysis of DAG. Little is known about DAG lipases. Two mammalian DAG lipase genes, DAGLα and -β, have been identified by a bioinformatics approach and characterized both biochemically and molecularly (Bisogno, 2003), and many proteins homologous to DAGα and -β have been identified across species. In the case of Drosophila, Huang (2004) has described a mutant, rolling blackout (rbo), which was suggested might be in a DAG lipase gene. The protein encoded by the rbo gene, however, shows little homology to the known mammalian DAG lipases. Moreover, conditional loss of the RBO protein leads to rapid depletion of DAG, the opposite of what one would expect if RBO catalyzes the hydrolysis of DAG. Furthermore, in the absence of previous activity, the receptor potential is normal in rbo mutants, making it unlikely that RBO has any direct involvement in the activation of TRP/TRPL channels (Huang, 2004). Other than rbo, no candidate DAG lipase that might function in phototransduction has been reported in any species (Leung, 2008).

This work reports on a Drosophila DAG lipase (DAGL) gene, inaE, identified from mutants that are defective in photoreceptor responses to light. The protein isoforms encoded by this gene show high sequence similarity to the two known mammalian DAGLs, exhibit DAGL activity in vitro, are highly expressed in photoreceptors, and have access to rhabdomeres. Genetic evidence suggests that the inaE-encoded DAGLs interact in vivo with the DAG generated in the phototransduction cascade. Analysis of mutants generated by imprecise excision of P element insertion in inaE show that no physiologically meaningful photoreceptor responses can be generated if inaE gene is severely impaired (Leung, 2008).

The inaE gene was identified by two ethylmethane sulfonate (EMS)-induced allelic mutants: N125 and P19. These mutants are characterized by their 'ina' (inactivation, no afterpotential) electroretinogram (ERG) phenotype. Wild-type flies, when placed on a white-eye (w) background, respond to a bright blue stimulus with a large response during light stimulus followed by a prolonged depolarizing afterpotential (PDA) after the light is turned off. A second blue stimulus elicits only a small response, originating from R7/8 photoreceptors, superposed on the PDA. By contrast, in ina mutants, the response begins to decay during stimulus (inactivation), and the decay continues after the stimulus. As a result, the PDA is greatly diminished in amplitude (no afterpotential). This phenotype can also be viewed as a mild form of the 'trp' phenotype displayed by strong mutants of the TRP channel gene. In trp mutants, the response to the first blue stimulus decays nearly to baseline during stimulus, and there is no PDA (Leung, 2008).

Moreover, responses of inaE mutants resemble those of trp in that they both display refractory properties. Following a response to the first stimulus, only very small responses can be elicited from trp until they recover over a period of minutes, while wild-type responses recover almost immediately. Likewise, inaE mutants exhibit a similar refractory period, although the degree and duration of response suppression are not as pronounced or prolonged as in trp (Leung, 2008).

In addition to the above similarities, inaE^N125 acts as a genetic enhancer of Trp^P365. Trp^P365 is a semidominant allele of trp, which causes constitutive activation of TRP channels and, as a result, massive photoreceptor degeneration from excessive Ca²⁺ influx. In Trp^P365 homozygotes, degeneration is already so advanced in 1- to 2-day-old flies that essentially no ERGs can be elicited. Trp^P365 heterozygotes exhibit a much milder phenotype and elicit ERGs of substantial amplitude at the same age. However, if inaE^N125 is introduced into the Trp^P365/+ background, the resulting phenotype is as severe as that of Trp^P365 homozygotes. The genetic enhancement of Trp^P365/+ by inaE^N125 and the basic similarity of ERG phenotypes between inaE and trp mutants led to the hypothesis that the protein products of these two genes may interact and/or subserve closely related functions (Leung, 2008).

The CG33174 gene had not been characterized previously, and its function was electronically inferred as 'triacylglycerol lipase (TAGL) activity'. However, the possibility is considered that the above annotation could simply reflect a dearth of information on DAGLs. The first two human DAGLs, DAGLα and -β, were cloned and characterized by Bisogno (2003) by a bioinformatic approach and shown to be sn-1 type DAGLs. Multiple alignment of INAE-A and INAE-D with DAGLα and -β revealed extensive sequence and domain conservations. All four proteins are predicted to have four transmembrane segments near the N-terminal region, and they all have a lipase_3 domain with a highly conserved serine active site. Overall sequence homology between INAE-D and the two human DAGLs is 39% identity and 56% similarity for DAGLα and 30% identity and 50% similarity for DAGLβ, respectively. In the lipase_3 domain, the sequence homology between INAE-A/D and the mammalian proteins rises to 60% and 45% identity and 73% and 63% similarity for DAGLα and DAGLβ, respectively (Leung, 2008).

To demonstrate that the INAE proteins have DAG lipase activity, the INAE-A and -D protein isoforms were expressed in E. coli and purified to >95% purity to carry out DAG lipase assays using 1-stearoyl-2-arachidonoyl-sn-glycerol as substrate, and the lipase assay products were analyzed by liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) for identification of hydrolysis products and kinetic studies (Leung, 2008).

LC-MS detected four products from the analysis of both INAE isoforms: two primary products, stearic acid (18:0) and 2-arachidonoyl glycerol (2-AG), and two minor products, arachidonic acid (20:4) and 1-stearoyl glycerol (1-SG), eluting at 6.3, 9.3, 5.8, and 8.7 min, respectively. Two primary products corresponded to hydrolysis at the sn-1 position of DAG substrate, and the two minor products corresponded to hydrolysis at the sn-2 position. Thus, in vitro, the two recombinant INAE isoforms are both DAG lipases highly preferential for hydrolysis at the sn-1 position, with the D form having much higher activity than the A form (Leung, 2008).

A growing body of evidence suggests that Drosophila phototransduction utilizes the DAG branch of the G protein-coupled, PLC_β-mediated signaling pathway). Although DAG lipase is expected to play a critical role in this pathway, no DAG lipase that could play such a role had been identified previously in Drosophila. This study reports on a DAG lipase identified from the Drosophila mutants, inaE. The inaE gene was found to encode two protein isoforms, INAE-A and INAE-D, by alternative splicing. Both of these proteins are highly homologous to the two previously identified mammalian sn-1 type DAG lipases, and in vitro DAG lipase assays of recombinant INAE-A and INAE-D showed that both are DAG lipases highly preferential for hydrolysis at the sn-1 position. Expression of the INAE protein is not restricted to the eye but occurs throughout the head, consistent with the finding that strong mutations in this gene are homozygous lethal. In photoreceptors, anti-INAE antibody labeling occurs as punctate staining scattered throughout the photoreceptor cytoplasm. Occasionally, some of the puncta are found within the rhabdomeres, indicating that some DAGL enters the rhabdomeres. Results of the norpA inaE double mutant study provide strong functional support for the above observation. In this study, the receptor potential disappears in an inaE allele-dependent manner -- the stronger the inaE allele in the double mutant, the more severe the double mutant phenotype. The allele dependence strongly suggests that the action of inaE-encoded DAGL is responsible for the observed double mutant phenotype. Furthermore, to affect the receptor potential phenotype, DAGL must act on the DAG generated by norpA-encoded PLC_β, and, for that to occur, DAGL must enter the rhabdomeres (Leung, 2008).

Because inaE mutations already available were all relatively mild, severe mutations were generated by imprecise excisions of a P element insertion in the inaE gene. These imprecise excision alleles were homozygous lethal and had to be studied as eye mosaics. Quantitative RT-PCR results showed that even the severest of these imprecise excision mutants, inaE^xl18, is not a null mutant and expresses RNA at ~25% of the normal level. This mutation profoundly affects the photoreceptor responses to light. If xl18 is placed on a norpA^H43 background to reduce the amount of DAG generated, the light stimulus generates no response at all. In xl18 flies themselves, a bright prolonged stimulus generates only a small response of slow kinetics that decays to baseline completely during the stimulus. This response most likely represents the residual DAGL activity in this severely affected mutant. As the severity of mutation progressively decreases in the xl series of mutants, the receptor potential phenotype returns to normal in an allele-dependent manner. Again, the inaE allele dependence strongly argues that the action of inaE-encoded DAGL is responsible for the observed change in the receptor potential phenotype. These results, taken together, suggest (1) that the production of DAG metabolite(s) through the action of the inaE-encoded DAGL is required for the generation of photoreceptor responses to light and (2) that, in the absence of the metabolite, DAG plays little direct role in the activation of channels. However, the identity of the excitatory molecule cannot be specified from this work. It could be one or more of the products generated by INAE, such as monoacylglycerol (2-AG) or stearic acid or even DAGL (INAE) itself (Leung, 2008).

While DAG may not have a direct role in channel activation, evidence was found suggesting that it may be important in regulating the action of the DAG metabolite that acts as an excitatory agent, although the evidence is still largely indirect. The ability of inaE^N125 to act as an enhancer of Trp^P365/+ seems to present a quandary when considered in relation to the results summarized above. If DAG has little or no direct role in channel activation, how does one explain the disappearance of the small response present in P365/+ when N125 is added to this background? A simple explanation for the phenomenon would be that DAG is excitatory to the channels and that adding N125 to the P365/+ background raised the level of DAG to make more channels to become constitutively active in N125;P365/+ than in P365/+. However, results of the experiment replacing N125 with a stronger inaE allele, xl18, in the N125;P365/+ double mutant run counter to this simple explanation. Replacing N125 with xl18 should have sharply raised the basal DAG level further in the double mutant. If DAG were excitatory, the resting potential should have depolarized even more than before the N125 replacement, and no receptor potential at all should have been obtained. Just the opposite results were obtained. A small but distinct receptor potential could be recorded from xl18;P365/+, and the resting potential has returned to the level in P365/+. These results are incompatible with the hypothesis that DAG is excitatory to the channel and instead provide another line of evidence for the conclusions summarized earlier (Leung, 2008).

However, the fact that a much more severe phenotype is obtained in N125;P365/+ than in P365/+ or xl18;P365/+ suggests that DAG may have a role in facilitating, enhancing, and orchestrating the action of the DAG metabolite that serves as the excitatory agent. This action of DAG would be more noticeable under conditions in which a sufficient amount of the excitatory product is produced, as in N125;P365/+ rather than in xl18;P365/+. A similar action of DAG can also be inferred from the norpA inaE double mutant studies. In this series of experiments, hypomorphic norpA mutation, H43, was used to restrict the amount of DAG generated both in the single and double mutants. The response obtained from H43 N125 is short in duration but has nearly the same maximum amplitude as the H43 response and much faster time course of rise than the H43 response. The shortness of response duration may be due to the fact that, under the conditions of this experiment (restricted DAG generation), the response cannot be sustained during a bright prolonged stimulus. This response arises as a result of DAGL activity because further reducing the DAGL activity (xl18 mutation) abolishes the response. However, the fact that adding N125 to the H43 background resulted in a response of faster time course may also be a manifestation of the enhancing and facilitatory effects of DAG on the excitatory agent. Speculating further, the facilitatory action of DAG might be in place to ensure that the excitation of channels is light regulated, because DAGL activity is not light regulated while the generation of DAG is (Leung, 2008).

Diacylglycerol lipase regulates lifespan and oxidative stress response by inversely modulating TOR signaling in Drosophila and C. elegans

Target of rapamycin (TOR) signaling is a nutrient-sensing pathway controlling metabolism and lifespan. Although TOR signaling can be activated by a metabolite of diacylglycerol (DAG), phosphatidic acid (PA), the precise genetic mechanism through which DAG metabolism influences lifespan remains unknown. DAG is metabolized to either PA via the action of DAG kinase or 2-arachidonoyl-sn-glycerol by diacylglycerol lipase (DAGL). This study reports that in Drosophila and Caenorhabditis elegans, overexpression of diacylglycerol lipase (DAGL/inaE/dagl-1) or knockdown of diacylglycerol kinase (DGK/rdgA/dgk-5) extends lifespan and enhances response to oxidative stress. Phosphorylated S6 kinase (p-S6K) levels are reduced following these manipulations, implying the involvement of TOR signaling. Conversely, DAGL/inaE/dagl-1 mutants exhibit shortened lifespan, reduced tolerance to oxidative stress, and elevated levels of p-S6K. Additional results from genetic interaction studies are consistent with the hypothesis that DAG metabolism interacts with TOR and S6K signaling to affect longevity and oxidative stress resistance. These findings highlight conserved metabolic and genetic pathways that regulate aging (Lin, 2014).

Search PubMed for articles about Drosophila inaE

Bisogno, T., et al. (2003). Cloning of the first sn1-DAG lipases points to the spatial and temporal regulation of endocannabinoid signaling in the brain. J. Cell Biol. 163: 463-468. PubMed ID: 14610053

Chyb, S., Raghu, P. and Hardie, R. C. (1999). Polyunsaturated fatty acids activate the Drosophila light-sensitive channels TRP and TRPL. Nature 397(6716): 255-9. PubMed ID: 9930700

Hardie, R. C., et al. (2002). Molecular basis of amplification in Drosophila phototransduction: roles for G protein, phospholipase C, and diacylglycerol kinase. Neuron 36: 689-701. PubMed ID: 12441057

Hardie, R. C. (2003). Regulation of TRP channels via lipid second messengers. Annu. Rev. Physiol. 65: 735-759. PubMed ID: 12560473

Huang, F. D., et al. (2004). Rolling blackout, a newly identified PIP2-DAG pathway lipase required for Drosophila phototransduction. Nat. Neurosci. 7: 1070-1078. PubMed ID: 15361878

Leung, H. T., Tseng-Crank, J., Kim, E., Mahapatra, C., Shino, S., Zhou, Y., An, L., Doerge, R. W. and Pak, W. L. (2008). DAG lipase activity is necessary for TRP channel regulation in Drosophila photoreceptors. Neuron 58(6): 884-96. PubMed ID: 18579079

Lin, Y. H., Chen, Y. C., Kao, T. Y., Lin, Y. C., Hsu, T. E., Wu, Y. C., Ja, W. W., Brummel, T. J., Kapahi, P., Yuh, C. H., Yu, L. K., Lin, Z. H., You, R. J., Jhong, Y. T. and Wang, H. D. (2014). Diacylglycerol lipase regulates lifespan and oxidative stress response by inversely modulating TOR signaling in Drosophila and C. elegans. Aging Cell [Epub ahead of print]. PubMed ID: 24889782

Raghu, P., et al. (2000). Constitutive activity of the light-sensitive channels TRP and TRPL in the Drosophila diacylglycerol kinase mutant, rdgA. Neuron 26(1): 169-79. PubMed ID: 10798401

date revised: 23 July 2014

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