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Rad, Gem/Kir family member 1: Biological Overview | References

For aversive olfactory memory in Drosophila, multiple components have been identified that exhibit different stabilities. Intermediate-term memory generated after single cycle conditioning is divided into anesthesia-sensitive memory (ASM) and anesthesia-resistant memory (ARM), with the latter being more stable. This study determined that the ASM and ARM pathways converged on the Rgk1 small GTPase and that the N-terminal domain-deleted Rgk1 was sufficient for ASM formation, whereas the full-length form was required for ARM formation. Rgk1 is specifically accumulated at the synaptic site of the Kenyon cells (KCs), the intrinsic neurons of the mushroom bodies (MBs), which play a pivotal role in olfactory memory formation. A higher than normal Rgk1 level enhanced memory retention, which is consistent with the result that Rgk1 suppressed Rac-dependent memory decay; these findings suggest that rgk1 bolsters ASM via the suppression of forgetting. It is proposed that Rgk1 plays a pivotal role in the regulation of memory stabilization by serving as a molecular node that resides at KC synapses, where the ASM and ARM pathway may interact (Murakami, 2017).

Drosophila olfactory learning and memory, in which an odor is associated with stimuli that induce innate responses such as aversion, has served as a useful model with which to elucidate the molecular basis of memory. Olfactory memory is divided into several temporal components and the intermediate-term memory (ITM) generated after single cycle conditioning is further classified into two distinct phases, anesthesia-sensitive memory (ASM) and anesthesia-resistant memory (ARM). Evidence has suggested that ASM and ARM are distinctly regulated at the neuronal level and at the molecular level (Murakami, 2017).

Mushroom bodies (MBs) represent the principal mediator of olfactory memory. Kenyon cells (KCs) are the intrinsic neurons of MBs, which are bilaterally located clusters of neurons that project anteriorly to form characteristic lobe structures and are a platform of MB-extrinsic neurons that project onto or out of the MBs. To elucidate the molecular mechanisms that underlie olfactory memory, screenings for MB-expressing genes have been a useful strategy. A technique used to examine gene expression in a small amount of tissue samples has enabled the investigation of the expression profile in MBs with a substantial dynamic range of expression levels and high sensitivity, thereby representing a promising approach with which to identify novel genes responsible for memory. This study deep sequenced RNA isolated from adult MBs and identified rgk1 as a KC-specific gene (Murakami, 2017).

The RGK protein family, for which Drosophila Rgk1 exhibits significant protein homology, belongs to the Ras-related small GTPase subfamily, which is composed of Kir/Gem, Rad, Rem, and Rem2. Their roles include the regulation of Ca2+ channel activity and the reorganization of cytoskeleton. Notably, mammalian REM2 is expressed in the brain and has been shown to be important for synaptogenesis, as well as activity-dependent dendritic complexity. These findings raise the possibility that RGK proteins may have a role in the synaptic plasticity that underlies memory formation. Drosophila has several genes that encode proteins homologous to the RGK family, including rgk1. Therefore, based on the ample resources available in Drosophila for the investigation of neuronal morphology and functions, Drosophila Rgk proteins will provide a good opportunity to elucidate the function of RGK family proteins (Murakami, 2017).

This study describes the analysis of Drosophila rgk1, which exhibited specific expression in KCs. Rgk1 accumulated at synaptic sites and was required for olfactory aversive memory, making the current study the first to demonstrate the role of an RGK family protein in behavioral plasticity. These data suggest that Rgk1 supports ASM via the suppression of Rac-dependent memory decay, whereas the N-terminal domain has a specific role in ARM formation. Together, these findings indicated that Rgk1 functions as a critical synaptic component that modulates the stability of olfactory memory (Murakami, 2017).

It is proposed that the ITM is genetically divided into three components: the rut-, dnc-, and rgk1-dependent pathways. The rut and dnc pathway act specifically for ASM and ARM, respectively, whereas rgk1 acts for both ASM and ARM, albeit partially. Consistent with this notion, it is noteworthy that the ASM and ARM pathways converge on Rgk1, yet the functional domains may be dissected; the full-length form of Rgk1 is required for ARM, whereas the molecule that lacks the N-terminal domain is capable of generating ASM, which suggests that the protein(s) required for ARM formation may interact with the N-terminal domain of Rgk1 (Murakami, 2017).

The data suggested that Rgk1 acts for both ASM and ARM, whereas the rgk1 deletion mutant, which was shown to be a protein null, exhibited only a partial reduction in ITM; these findings imply that Rgk1 regulates an aspect of each memory component. This idea may be explained by the expression pattern of Rgk1. Rgk1 exhibited exclusive expression and cell-type specificity in the KCs, whereas the memory components have been shown to be regulated by the neuronal network spread outside of the MBs and are encoded by multiple neuronal populations. For example, two parallel pathways exist for ARM and ASM is modulated, not only by MB-extrinsic neurons, but also by the ellipsoid body that localizes outside of the MBs. dnc-dependent ARM requires antennal lobe local neurons and octopamine-dependent ARM requires α'/β' KCs, in neither of which was Rgk1 detected. Therefore, Rgk1 may support a specific part of memory components that exists in a subset of KCs (Murakami, 2017).

The specific expression of Rgk1 in KCs suggests its dedicated role in MB function. Rgk1 exhibited cell-type specificity in KCs from anatomical and functional points of view. Rgk1 is strongly expressed in α/β and γ KCs and weakly expressed in α'/β' KCs and the expression of the rgk1-sh transgene in α/β and γ KCs was sufficient to disrupt memory. Several genes required for memory formation have been shown to be expressed preferentially in the KCs and the notable genes include dunce, rutabaga, and DC0. Although a recent study in KC dendrites showed that the modulation of neurotransmission into the KCs affects memory strength, KC synapses are thought to be the site in which memory is formed and stored. The current analyses with immunostaining and GFP fusion transgenes indicated that Rgk1 is localized to synaptic sites of the KC axons, which raises the possibility that Rgk1 may regulate the synaptic plasticity that underlies olfactory memory. Among the RGK family proteins, Rem2 is highly expressed in the CNS and regulates synapse development through interactions with 14-3-3 proteins, which have been shown to be localized to synapses and are required for hippocampal long-term potentiation and associative learning and memory. In Drosophila, 14-3-3Ζ is enriched in the MBs and is required for olfactory memory. In addition, the C-terminal region of Drosophila Rgk1 contains serine and threonine residues that exhibit homology to binding sites for 14-3-3 proteins in mammalian RGK proteins. Therefore, Rgk1 and 14-3-3Ζ may act together in the synaptic plasticity that underlies olfactory memory (Murakami, 2017).

The roles of RGK family proteins in neuronal functions have been investigated extensively. The current data, when combined with the accumulated data on the function of RGK family proteins, provide novel insights into the mechanism that governs two distinct intermediate-term memories, ASM and ARM. Regarding the regulation of ASM, the data showed that Rgk1 suppressed the forgetting that was facilitated by Rac. Rac is a major regulator of cytoskeletal remodeling. Similarly, mammalian RGK proteins participate in the regulation of cell shape through the regulation of actin and microtubule remodeling. Rgk1 may affect Rac activity indirectly by sharing an event in which Rac also participates because there have been no reports showing that RGK proteins regulate Rac activity directly; further, it was determined that rgk1 transgene expression did not affect the projection defect of KC axons caused by RacV12 induction during development. Therefore, it is suggested that Rgk1 signaling and Rac signaling may merge at the level of downstream effectors in the regulation of forgetting. A member of the mammalian RGK1 proteins, Gem, has been shown to regulate Rho GTPase signaling through interactions with Ezrin, Gimp, and Rho kinase. Rho kinase is a central effector for Rho GTPases and has been shown to phosphorylate LIM-kinase. In Drosophila, the Rho-kinase ortholog DRok has been shown to interact with LIM-kinase. Furthermore, Rac regulates actin reorganization through LIM kinase and cofilin and the PAK/LIM-kinase/cofilin pathway has been postulated to be critical in the regulation of memory decay by Rac. It was shown recently that Scribble scaffolds a signalosome consisting of Rac, Pak3, and Cofilin, which also regulates memory decay. Therefore, Rgk1 may counteract the consequence of Rac activity (i.e., memory decay) through the suppression of the Rho-kinase/LIM-kinase pathway. DRok is a potential candidate for further investigation of the molecular mechanism in which Rgk1 acts to regulate memory decay (Murakami, 2017).

The data indicated that Rgk1 is required for ARM in addition to ASM. It has been shown that Synapsin and Brp specifically regulate ASM and ARM, respectively. The functions of Synapsin and Brp may be differentiated in a synapse by regulating distinct modes of neurotransmission. The exact mechanism has not been identified for this hypothesis; however, the regulation of voltage-gated calcium channels may be one of the key factors that modulate the neurotransmission. Voltage-gated calcium channels are activated by membrane depolarization and the subsequent Ca2+ increase triggers synaptic vesicle release. The regulation of voltage-gated calcium channels has been shown to be important in memory; a β-subunit of voltage-dependent Ca2+ channels, Cavβ3, negatively regulates memory in rodents. Importantly, Brp regulates the clustering of Ca2+ channels at the active zone. Moreover, it has been demonstrated extensively that mammalian RGK family proteins regulate voltage-gated calcium channels. Kir/Gem and Rem2 interact with the Ca2+ channel β-subunit and regulate Ca2+ channel activity. In addition, the ability to regulate Ca2+ channels has been shown to be conserved in Drosophila Rgk1 (Puhl, 2014). Therefore, both Brp and Rgk1 may regulate ARM through the regulation of calcium channels, the former through the regulation of their assembly and the latter through the direct regulation of their activity. The finding that Rgk1 localized to the synaptic site and colocalized with Brp lends plausibility to the scenario that Rgk1 regulates voltage-gated calcium channels at the active zone (Murakami, 2017).

Several memory genes identified in Drosophila, including rutabaga, PKA-R, and CREB, have homologous genes that have been shown to regulate behavioral plasticity in other species. The identification of Drosophila rgk1 as a novel memory gene raises the possibility for another conserved mechanism that governs memory. There is limited research regarding the role of RGK proteins at the behavioral level in other species; however, the extensively documented functions of RGK proteins with respect to the regulation of neuronal functions, combined with the data presented in this study regarding Drosophila Rgk1, raise the possibility of an evolutionally conserved function for RGK family proteins in memory (Murakami, 2017).

Ancient origins of RGK protein function: modulation of voltage-gated calcium channels preceded the protostome and deuterostome split

RGK proteins, Gem, Rad, Rem1, and Rem2, are members of the Ras superfamily of small GTP-binding proteins that interact with Ca2+ channel beta subunits to modify voltage-gated Ca2+ channel function. In addition, RGK proteins affect several cellular processes such as cytoskeletal rearrangement, neuronal dendritic complexity, and synapse formation. To probe the phylogenetic origins of RGK protein-Ca2+ channel interactions, this study identified potential RGK-like protein homologs in genomes for genetically diverse organisms from both the deuterostome and protostome animal superphyla. RGK-like protein homologs cloned from Danio rerio (zebrafish) and Drosophila melanogaster (fruit flies) expressed in mammalian sympathetic neurons decreased Ca2+ current density as reported for expression of mammalian RGK proteins. Sequence alignments from evolutionarily diverse organisms spanning the protostome/deuterostome divide revealed conservation of residues within the RGK G-domain involved in RGK protein--Cavbeta subunit interaction. In addition, the C-terminal eleven residues were highly conserved and constituted a signature sequence unique to RGK proteins but of unknown function. Taken together, these data suggest that RGK proteins, and the ability to modify Ca2+ channel function, arose from an ancestor predating the protostomes split from deuterostomes approximately 550 million years ago (Puhl, 2014).

Search PubMed for articles about Drosophila Rgk1

Murakami, S., Minami-Ohtsubo, M., Nakato, R., Shirahige, K. and Tabata, T. (2017). Two components of aversive memory in Drosophila, anesthesia-sensitive and anesthesia-resistant memory, require distinct domains within the Rgk1 small GTPase. J Neurosci 37(22):5496-5510. PubMed ID: 28416593

Puhl, H. L., Lu, V. B., Won, Y. J., Sasson, Y., Hirsch, J. A., Ono, F. and Ikeda, S. R. (2014). Ancient origins of RGK protein function: modulation of voltage-gated calcium channels preceded the protostome and deuterostome split. PLoS One 9(7): e100694. PubMed ID: 24992013

date revised: 12 December 2018

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