InteractiveFly: GeneBrief

methuselah-like 1: Biological Overview | References

Injection of RasV12 oncogenic cells (OCs) into adult male flies induces a strong transcriptomic response in the host flies featuring in particular genes encoding bona fide G coupled proteins, among which the gene for methuselah like 1 is prominent. The injection is followed after a 3-d lag period, by the proliferation of the oncogenic cells. It was hypothesized that through the product of mthl1 the host might control, at least in part, this proliferation as a defense reaction. Through a combination of genetic manipulations of the mthl1 gene (loss of function and overexpression of mthl1), this study documented that indeed this gene has an antiproliferative effect. Parallel injections of primary embryonic Drosophila cells or of various microbes do not exhibit this effect. It was further shown that mthl1 controls the expression of a large number of genes coding for chemoreceptors and genes implicated in regulation of development. Of great potential interest is the observation that the expression of the mouse gene coding for the adhesion G-protein-coupled receptor E1 (Adgre1, also known as F4/80), a potential mammalian homologue of mthl1, is significantly induced by B16-F10 melanoma cell inoculation 3 d postinjection in both the bone marrow and spleen (nests of immature and mature myeloid-derived immune cells), respectively. This observation is compatible with a role of this GPCR in the early response to injected tumor cells in mice (Chen, 2023).

Invertebrates have appeared several hundred millions of years before vertebrates and are estimated to make up some 95% of animal species on earth at present, as compared to 5% for extant vertebrates. They have been confronted since their appearance to an enormous variety of potential microbial aggressors, many of which are still present today. The antimicrobial defense mechanisms of insects have attracted increasing interest over the last decades and many laboratories worldwide have engaged in studies regarding the cellular and molecular basis of their highly efficient defense reactions. Several invertebrate species from various phyletic groups have yielded significant data in this respect. In particular, the genetically tractable Drosophila has proved extremely valuable for these studies. Over the years, it has thus become apparent that insects rely only on the innate arm of immune defenses to fight microbes and are devoid of the adaptive arm with its hallmark of immune memory, the basis for vaccination in humans. Significantly, at the end of the 1990s it was understood that innate immunity in vertebrates and invertebrates shared many similarities in terms of receptors of microbial aggressors and subsequent activations of intracellular signaling cascades leading to transcription of genes encoding defense proteins, namely antimicrobial peptides, to oppose the invaders (Chen, 2023).

It has been known for nearly a century that Drosophila, like many invertebrates, can develop tumors and a vast number of investigations have been devoted to the genetic origins of this process. In contrast, relatively few studies have addressed the question of the recognition of such tumors by the flies and their potential responses to these noninfectious insults. A series of investigations has recently been established to decipher the potential recognition mechanisms of tumor cells by host flies and the subsequent molecular and cellular reactions (Chen, 2023).

To simplify an experimental approach, the first studies were based on a model of injections of OCs into adult males (to avoid as much as possible unwanted interferences with developmental regulations in larvae/pupae and with the process of vitellogenesis in female adults). It is noted that the injected cells did not proliferate during the first 3 d postinjections (p.i.). After this apparent lag period, the cell numbers increased markedly. Between day 5 and day 11, a massive proliferation occurred. Until finally, half of the experimental flies had succumbed on day 11. In parallel, it is noted that the injections of the OCs induced an early (day 3 p.i.) remarkably strong transcriptomic response in the host flies. Unexpectedly, this transcriptomic response included over one hundred genes encoding chemoreceptors of various families, among which 12 are G-protein-coupled receptor (GPCR) family members. Of note, these experiments confirmed that the kinetics of induction and the identities of the induced genes differed markedly from the responses generated by parallel injections of microbes (Chen, 2023).

This study has undertaken a series of functional studies on the roles of the genes induced during the early stages following the injection of oncogenic cells. Focus was placed on chemoreceptor genes of the GPCR family induced in the model during the early period up to the massive transcriptomic wave around day 3 p.i. In particular, it is reported that one of the strongest-induced GPCR gene in in this study belongs to a family of previously described fly genes, which can affect (namely, but not solely) the life span negatively, and was termed by its discoverer S. Benzer for this reason, methuselah (mth) (Lin, 1997) in reference to the biblical figure Methuselah reported to have lived up to 996 years. It is now known that Drosophila mth belongs to a family of 16 genes (mth and mth like 1 to 15). Of great interest to this long-term project is the fact that similar genes were discovered in mammals around the same period and shown to play roles as adhesion molecules in various settings. The sequence similarity is particularly interesting between mthl1 and adhesion G-protein-coupled receptor E1 (Adgre1). This paper centers in on the functional analysis on the mthl1 gene - whose expression in experimental flies was one of the strongest after injection of OCs. Loss-of-function and gain-of-function mutants will be considered in the context of the early host response to the injection of oncogenic cells. Data is included that was obtained when following the expression of Adgre1 after inoculation of cancer cells (melanoma B16-F10) into C57/BL6 mice, which support the idea of similarities between the two models in the present context (Chen, 2023).

The results presented here call for several comments, in particular regarding the following points: (1) In a previous study, it was noticed that the proliferation of injected OCs is very limited during day 0 to day 4 p.i., (referred to as lag period), yet there is a strong response in terms of transcriptomic activity at day 3 p.i. In this study, we observed that in mthl1 LOF flies, the injection resulted in a rapid and significant increase in proliferation of injected OCs in the flies. In keeping with this result, overexpression of mthl1 suppressed significantly the proliferation of the injected OCs. These results indicate that MTHL1 is a major regulator of the proliferation of OCs and point to a clear role of this receptor in a targeted anti-cancer defense reaction. At this stage, it cannot be excluded that other factors contribute to this antiproliferative role of mthl1 (Chen, 2023).

(2) At the later time point, this mthl1-dependent antiproliferative effect is partially overcome, most likely by an important change in the transcriptomic profile of injected OCs as documented in a previous study reporting the transcriptomic profiles of injected OCs had evolved for 11 d in the experimental flies following their injection on day 0. Additional data are required to establish this hypothesis more firmly, which is a present priorities (Chen, 2023).

(3) Whereas OC injection clearly induces mthl1 expression in the Drosophila host, which in turn represses the proliferation of the injected OCs, the same effects are not observed when primary Drosophila ECs or pathogenic microbes (namely viruses and gram-positive and -negative bacteria) are injected into flies. This indicates that the ligand(s) of mthl1 come(s) either directly from the injected OCs or from so far unspecified modifications resulting possibly from distinct immunopathological effects induced by the injection of the OCs (Chen, 2023).

(4) The transcriptomic analysis of mthl1-dependent genes interestingly reveals that many (but not all) chemoreceptors induced by injected OCs in wild-type flies are positively regulated by mthl1, point to a chemoreceptors cascade. Their functions need to be further explored, which will of course imply identifications of their respective ligands (Chen, 2023).

(5) In parallel, mthl1 represses the expressions of genes from several important developmental pathways, namely dpp, hh, wingless (wg), Notch (N), etc. As a reminder, the OCs originate from embryonic Drosophila cells, in which these developmental pathways are active and crucial for the growth and proliferation of these cells. Repression of these genes in the injected OCs by the host is thus in keeping with the antiproliferative effect, which was observed during the lag period (Chen, 2023).

(6) There are seven members from adhesion GPCRs family in mammals predicted to be the homologues of mthl1 (according to the sequence similarities as noted in FlyBase/NCBI). Among them, Adgre1 has the highest similarity with mthl1. Adgre1 encodes for F4/80 antigen, which is a widely used marker for monocyte macrophages. It is also found to be expressed in some myeloid-derived cells in mice (eosinophils, monocytes, macrophages, and dendritic cells) and other mammals, namely pig, human, etc. It is reported to be involved in myeloid-derived immune cells' development and defense reactions. However, the roles of mammalian adhesion GPCRs in response to tumor cells have been poorly investigated. This study found that the expression of Adgre1 in both bone marrow and spleen (nests of immature and mature myeloid-derived immune cells, respectively) is significantly induced by melanoma cell inoculation 3 days p.i. This suggests that one or several adhesion GPCRs may be involved in the early response to injected tumor cells in mice. This raises the exciting hypothesis that innate immune defenses against cancerous cells in flies and mammals share some of their characteristics. More specifically, the injection of tumor cells might increase the population of myeloid-derived macrophages in mice. If validated by future studies, this hypothesis would extend the observations of stringent parallelisms between innate defenses against microbes in flies and mammals documented in earlier studies in the field (Chen, 2023).

Structure-function analysis of beta-arrestin Kurtz reveals a critical role of receptor interactions in downregulation of GPCR signaling in vivo

Arrestins control signaling via the G protein coupled receptors (GPCRs), serving as both signal terminators and transducers. Previous studies identified several structural elements in arrestins that contribute to their functions as GPCR regulators. However, the importance of these elements in vivo is unclear, and the developmental roles of arrestins are not well understood. An in vivo structure-function analysis of Kurtz (Krz), the single ortholog of mammalian beta-arrestins in the Drosophila genome, was carried out. A combination of Krz mutations affecting the GPCR-phosphosensing and receptor core-binding ('finger loop') functions (Krz-KKVL/A) resulted in a complete loss of Krz activity during development. Endosome recruitment and bioluminescence resonance energy transfer (BRET) assays revealed that the KKVL/A mutations abolished the GPCR-binding ability of Krz. The isolated 'finger loop' mutation (Krz-VL/A), while having a negligible effect on GPCR internalization, severely affected Krz function, suggesting that tight receptor interactions are necessary for proper termination of signaling in vivo. Genetic analysis as well as live imaging demonstrated that mutations in Krz led to hyperactivity of the GPCR Mist (also known as Mthl1), which is activated by its ligand Folded gastrulation (Fog) and is responsible for cellular contractility and epithelial morphogenesis. Krz mutations affected two developmental events that are under the control of Fog-Mist signaling: gastrulation and morphogenesis of the wing. Overall, these data reveal the functional importance in vivo of direct beta-arrestin/GPCR binding, which is mediated by the recognition of the phosphorylated receptor tail and receptor core interaction. These Krz-GPCR interactions are critical for setting the correct level of Fog-Mist signaling during epithelial morphogenesis (Chai, 2019).

This study reveals the developmentally important structural elements in the Drosophila β-arrestin Krz. Several lines of evidence, obtained in vivo and in cultured Drosophila cells, support a view that the residues most critical for Krz function during development include the phosphate-sensing region (disrupted by the KK/A mutation) and the finger loop (disrupted by the VL/A mutation). These motifs are engaged in direct interactions with the phosphorylated GPCR tail and the receptor core region, respectively, and both contribute to the tight binding of β-arrestins to the receptor. Since such interactions mediate efficient uncoupling of the receptor from G proteins, it appears that the primary function of Krz in development is to inhibit GPCR signaling via receptor desensitization (Chai, 2019).

The combined mutation Krz-KKVL/A resulted in a complete inactivation of Krz as a developmental regulator, however the disruption of the two key phosphate-sensing residues together with an additional phosphate-binding residue (KKR/A), leaving the finger loop intact, was not sufficient to eliminate Krz zygotic function. Structural studies suggest that the G proteins and the finger loop of arrestins make contacts with the same region in GPCRs. Therefore, the binding of arrestins' finger loop to the receptor directly interferes with the GPCR/G protein interactions, and mutations of this motif are expected to disrupt the ability of arrestins to uncouple GPCRs from G proteins. In support of this view, this study found that embryos from Krz-VL/A-rescued females showed gastrulation defects and cuticular phenotypes that were similar to the ones observed for krz¹, the strongest loss of function allele of krz. These findings suggest that the finger loop region of Krz is critical for its in vivo functions. It is noted that embryos obtained from Krz-KK/A-rescued females aborted development before cellularization, which is a phenotype that is even stronger than that observed in maternal krz¹ mutants. The KK/A mutation may thus have a dominant-negative effect, possibly due to a global hyperactivity and dysregulation of multiple GPCRs (Chai, 2019).

A major GPCR system involved in early Drosophila embryogenesis is the Fog signaling pathway. After several steps of signal transduction, activation of Fog signaling culminates in phosphorylation of Sqh, which controls acto-myosin contractility and mediates apical constrictions. Setting a proper level of cellular contractility is required for gastrulation movements. Consistent with a recent report, this study found that loss of krz resulted in excessive accumulation of Sqh-GFP in the mid-apical region of ventral cells, indicating overactivation of Fog signaling. Live imaging showed that mid-apical accumulation of Sqh-GFP persists much longer in krz maternal mutants than in wild type embryos, resulting in a stalled and aberrant gastrulation. It is noted that the area of cells undergoing apical constriction was wider in krz¹ maternal mutants, which may be explained in part by ectopic activity of the Toll signaling pathway, that may lead to an expansion of Fog and Mist expression domains downstream of Twist activation. However, medial-apical Sqh-GFP localization persisted in krz¹ mutants even in cells along the ventral midline, suggesting that Krz is required to limit the activity of the Fog pathway within the normal domain of its activation. In support of this view, maternal knockdown of krz by RNAi did not result in an expansion of Twist expression, yet led to abnormal mid-apical accumulation of Sqh (Chai, 2019).

Fog signals through its receptors Mist and Smog, and Krz may control Smog signaling at the level of endocytosis. Analyses of Krz mutants that were expected to disrupt clathrin and AP2 interactions (Krz-ΔLIQLD and Krz-F/A, respectively) showed that neither of these mutations affected Krz functions. Both Krz variants rescued krz¹ homozygotes to adulthood, did not have maternal effect, and internalized into endosomes with CrzR upon ligand activation, though Krz-Δ;LIQLD and the double Krz-Δ;LIQLD ​+ ​F/A mutant did show a mild reduction in the percentage of cells containing endosomes. It is possible that Krz relies on other structural elements to mediate GPCR endocytosis that were not included in this analysis, such as a short motif present in Krz that resembles the second clathrin-binding region in the long splice variant of β-arrestin-1. However, it is also possible that the endocytic function of Krz is secondary to the role of direct receptor engagement. Consistent with this view, it was found that mutation of the finger loop (Krz-VL/A) resulted in a strong reduction in Krz function, without affecting endocytosis. Deletion of the finger loop region in rat β-arrestin-1 also did not affect its ability to internalize GPCRs but impaired its ability to uncouple the receptor from G proteins. These results suggest that Krz-mediated attenuation of GPCR and G protein signaling via direct receptor interactions is critical during early development in Drosophila, though receptor internalization may also be involved as an additional regulatory step (Chai, 2019).

In addition to controlling gastrulation, the Fog-Mist signaling is involved in the development of the wing, and the data show that Krz-mediated regulation of this pathway also occurs in this tissue. The effects of the phospho-sensing and finger loop mutations were even more pronounced in the wing, as neither Krz-KK/A nor Krz-VL/A were capable of effectively suppressing the gain of function Fog phenotype. Thus, Krz controls Fog-Mist signaling in the embryo and in the wing, and perhaps in other tissues where this pathway guides epithelial morphogenesis. It was previously reported that loss of krz had no significant effect on wing development. It is possible that the discrepancy with the current results was due to the use of the specific GAL4 drivers used in that study (638-GAL4 and nub-GAL4), that may be expressed in a more restricted pattern or at a lower level than the MS1096-GAL4 driver that this study used. Consistent with the observation of Krz requirement in wing development, homozygous mutant krz¹ flies that are rescued with the elavC155-GAL4 driver, which is primarily expressed in the nervous system, have abnormal wings - a result that this study confirmed (Chai, 2019).

Phosphorylation of GPCRs by G protein-coupled receptor kinases (GRKs in mammals, Gprk2 in Drosophila) is a required step for the recognition of the activated receptor by arrestins. Previously, mutations in Gprk2 were shown to result in gastrulation defects that are remarkably similar to the phenotypes observed in krz maternal mutants, suggesting that phosphorylation of GPCRs by Gprk2 is required for the binding of Krz to the receptors. In support of this view, the Krz-KK/A mutant, which disrupts the phosphate-sensing residues, lost its ability to suppress the Fog overexpression phenotype in the wing and exhibited a strong maternal effect. This study also found that overexpression of Gprk2 itself could suppress overactive Fog signaling in the wing. Gprk2 and Krz thus operate in concert to inhibit GPCR signaling (Chai, 2019).

While Krz has been implicated in the regulation of several signaling pathways, this study highlights the developmental importance of Krz in controlling its prototypical targets, GPCRs. In all contexts examined in this study, Krz plays an inhibitory role, which is likely mediated by direct engagement of the activated GPCRs via the two-part recognition of the phosphorylated receptor tail and its core. It will be of interest to examine how Krz coordinates its involvement in the various pathways active in the early embryo, such as the GPCR, ERK, and Toll signaling pathways, and whether these pathways are differentially affected by the mutations examined in this study. Of note, it was found that the pre-activating mutation Krz-R/E did not affect Krz interaction with the Toll pathway regulator Cactus, but did increase the association of Krz with ERK. With regard to ERK signaling, it was previously shown that Krz inhibits ERK activation downstream of receptor tyrosine kinases in Drosophila development. One question for future studies is to determine whether Krz is involved in the positive signaling events downstream of GPCRs, given the recent interest in developing G protein- and β-arrestin-biased ligands that modulate ERK activation and can increase the specificity and efficacy of GPCR-directed therapies. Rescue of krz¹ homozygous animals by both human β-arrestins shows significant functional conservation and suggests that Drosophila Krz can serve as a platform for modeling mammalian β-arrestin functions (Chai, 2019).

This work has used the Drosophila β-arrestin Kurtz (Krz) to identify conserved structural elements in the β-arrestin molecule that are functionally important in vivo. Some of the previously identified elements appear to be dispensable. Nonetheless, it was revealed that two regions in Krz are critical for its activity: a phosphate sensing motif which interacts with the phosphorylated GPCR tail, and a 'finger loop' region that directly contacts the GPCR core. The finger loop mutation (VL/A) is notable because it severely affected Krz function without having a significant effect on its ability to internalize a GPCR in endosomes. This study showed that GPCR-binding Krz mutations disrupt its ability to limit the activity of the Fog-Mist signaling pathway that plays a key role during epithelial morphogenesis events, such as embryo gastrulation and folding of the wing epithelium. These studies thus uncovered the structural motifs in β-arrestins that are critical for their developmental functions as GPCR regulators in vivo (Chai, 2019).

Methuselah/Methuselah-like G protein-coupled receptors constitute an ancient metazoan gene family

Inconsistent conclusions have been drawn regarding the phylogenetic age of the Methuselah/Methuselah-like (Mth/Mthl) gene family of G protein-coupled receptors, the founding member of which regulates development and lifespan in Drosophila. This study reports the results from a targeted homolog search of 39 holozoan genomes and phylogenetic analysis of the conserved seven transmembrane domain. It was found that the Mth/Mthl gene family is ancient, has experienced numerous extinction and expansion events during metazoan evolution, and acquired the current definition of the Methuselah ectodomain during its exceptional expansion in arthropods. In addition, Mthl1, Mthl5, Mthl14, and Mthl15 are the oldest Mth/Mthl gene family paralogs in Drosophila. Future studies of these genes have the potential to define ancestral functions of the Mth/Mthl gene family (de Mendoza, 2016).

Search PubMed for articles about Drosophila Hthl1

Chai, F., Xu, W., Musoke, T., Tarabelsi, G., Assaad, S., Freedman, J., Peterson, R., Piotrowska, K., Byrnes, J., Rogers, S. and Veraksa, A. (2019). Structure-function analysis of beta-arrestin Kurtz reveals a critical role of receptor interactions in downregulation of GPCR signaling in vivo. Dev Biol. PubMed ID: 31325455

Chen, D., Lan, X., Huang, X., Huang, J., Zhou, X., Liu, J. and Hoffmann, J. A. (2023). mthl1, a potential Drosophila homologue of mammalian adhesion GPCRs, is involved in antitumor reactions to injected oncogenic cells in flies. Proc Natl Acad Sci U S A 120(30): e2303462120. PubMed ID: 37459549

de Mendoza, A., Jones, J.W. and Friedrich, M. (2016). Methuselah/Methuselah-like G protein-coupled receptors constitute an ancient metazoan gene family. Sci Rep 6: 21801. PubMed ID: 26915348

Lin, Y.J., Benzer, S. (1997). Lifespan extension mutants in Drosophila. Doe, Hall, 1997 : 116.

date revised: 5 December 2023

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