InteractiveFly: GeneBrief

kurtz: Biological Overview | References


Gene name - kurtz

Synonyms -

Cytological map position- 100E3-100E3

Function - signaling

Keywords - non-visual β-arrestin, GPCR desensitization, Hedgehog pathway, Notch pathway, MAPK signaling and Toll signaling, Exploratory activity, olfactory sensitivity

Symbol - krz

FlyBase ID: FBgn0040206

Genetic map position - 3R:27,872,924..27,877,711 [-]

Classification - arrestin

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Zhang, J., Liu, Y., Jiang, K. and Jia, J. (2017). SUMO regulates the activity of Smoothened and Costal-2 in Drosophila Hedgehog signaling. Sci Rep 7: 42749. PubMed ID: 28195188
Summary:
In Hedgehog (Hh) signaling, the GPCR-family protein Smoothened (Smo) acts as a signal transducer that is regulated by phosphorylation and ubiquitination, which ultimately change the cell surface accumulation of Smo. However, it is not clear whether Smo is regulated by other post-translational modifications, such as sumoylation. This study demonstrates that knockdown of the small ubiquitin-related modifier (SUMO) pathway components Ubc9 (a SUMO-conjugating enzyme E2), PIAS (a SUMO-protein ligase E3), and Smt3 (the SUMO isoform in Drosophila) by RNAi prevents Smo accumulation and alters Smo activity in the wing. Hh-induced-sumoylation stabilizes Smo, whereas desumoylation by Ulp1 destabilizes Smo in a phosphorylation independent manner. Mechanistically, excessive Krz, the Drosophila β-arrestin 2, inhibits Smo sumoylation and prevents Smo accumulation through Krz regulatory domain. Krz likely facilitates the interaction between Smo and Ulp1 because knockdown of Krz by RNAi attenuates Smo-Ulp1 interaction. Finally, Cos2 is also sumoylated, which counteracts its inhibitory role on Smo accumulation in the wing. Taken together, these results uncover a novel mechanism for Smo activation by sumoylation that is regulated by Hh and Smo interacting proteins.
BIOLOGICAL OVERVIEW

The non-visual β-arrestins are cytosolic proteins highly conserved across species that participate in a variety of signalling events, including plasma membrane receptor degradation, recycling, and signalling, and that can also act as scaffolding for kinases such as MAPK and Akt/PI3K. In Drosophila, there is only a single non-visual β-arrestin, encoded by kurtz, whose function is essential for neuronal activity. This study addressed the participation of Kurtz in signalling during the development of the imaginal discs, epithelial tissues requiring the activity of the Hedgehog, Wingless, EGFR, Notch, Insulin, and TGFβ pathways. Surprisingly, it was found that the complete elimination of kurtz by genetic techniques has no major consequences in imaginal cells. In contrast, the over-expression of Kurtz in the wing disc causes a phenotype identical to the loss of Hedgehog signalling and prevents the expression of Hedgehog targets in the corresponding wing discs. The mechanism by which Kurtz antagonises Hedgehog signalling is to promote Smoothened internalization and degradation in a clathrin- and proteosomal-dependent manner. Intriguingly, the effects of Kurtz on Smoothened are independent of Gprk2 activity and of the activation state of the receptor. These results suggest fundamental differences in the molecular mechanisms regulating receptor turnover and signalling in vertebrates and invertebrates, and they could provide important insights into divergent evolution of Hedgehog signalling in these organisms (Molnar, 2011).

G-protein coupled receptors (GPCRs) are seven-transmembrane proteins that play critical roles during development and in the regulation of cellular physiology. GPCRs constitute the largest superfamily of cell membrane receptors. The major GPCR regulatory pathway involves phosphorylation of agonist-activated receptors by G protein-coupled receptor kinases (GRKs), followed by binding of the cytosolic arrestin proteins. This interaction prevents the receptor from activating additional G proteins in a process known as desensitization. GRKs and β-arrestins also participate in signal propagation by recruiting additional proteins to the receptor complex. Thus, the GRK/β-arrestin pathway facilitates receptor internalization from the cell surface through clathrin-coated pits, and this leads to numerous physiological outcomes, including receptor degradation, receptor recycling and the activation of distinct downstream signalling events. Finally, more recent evidence suggest a role for β-arrestins in signalling by other families of cellular receptors, including receptor tyrosine kinase (RTKs), non-classical 7TMRs like Smoothened and Frizzled, Notch and TGFβ receptors, and also by downstream kinases such as MAPK and Akt/PI3K (Molnar, 2011).

The arrestin family is divided in two classes: the visual arrestins (arrestin 1 and 4), which are located almost exclusively in photoreceptor cells, and the non-visual β-arrestins 1 and 2 (also named arrestin 2 and 3, respectively), which are ubiquitously distributed (Lefkowitz, 2005). These proteins are closely related and their sequence is highly conserved across species (Gurevich, 2004). In Drosophila melanogaster there is only a single non-visual β-arrestin, encoded by kurtz (krz), which function is essential for development, survival and neural function (Roman, 2000; Ge, 2006; Liu, 2007; Johnson, 2008). In addition, the gene CG32683 encodes a related protein that presents some homology with β-arrestins, but lacks the clathrin-binding domain. The GRK family includes seven members in humans (GRK1-7) and two components in flies (Gprk1 and Gprk2). Gprk1 modulates the amplitude of the visual response, acting as a Rhodopsin kinase, whereas Gprk2 regulates the level of cAMP during Drosophila oogenesis. In addition, Gprk2 and Gprk1 play a key role in the regulation of the Hedgehog (Hh) signal transduction pathway, where they seem to phosphorylate and activate the seven-pass transmembrane protein Smoothened (Smo). The β-arrestin Krz has also been involved in the regulation of Notch signalling, promoting the formation of a trimeric Notch-Deltex-Krz complex that mediates the degradation of the Notch receptor in an ubiquitination-dependent pathway (Mukherjee, 2005), reminiscent of β-arrestin-mediated ubiquitination of other canonical GPCRs. More recently, Krz has also been implicated in the regulation of Smo accumulation (Cheng, 2010) and ERK phosphorylation (Tipping, 2010). Because Krz is the unique β-arrestin present in Drosophila, it is likely that the protein has additional functions in the modulation of other signalling pathways (Molnar, 2011).

To address the participation of Krz in signalling events, its function during the development of the imaginal discs, the epithelial layers that give rise to the adult structures of the fly, was studied. Imaginal discs are very convenient model systems to study the activity of signalling pathways in vivo, because their development is under the regulation of the Hh, Wingless, EGFR, Notch, Insulin and TGFβ pathways. In this manner, the response of these epithelia to the manipulation of Krz levels using genetic variants is a key diagnostic to identify the functional requirements of this protein in signalling during imaginal development. Surprisingly, considering the key roles identified for vertebrate non-visual arrestins, this study found that the complete elimination of Krz in imaginal cells has no major consequences during imaginal development. Thus, and as claimed previously (Roman, 2000), krz mutant flies are morphologically normal. In contrast, the over-expression of Krz in the wing causes a phenotype identical to the loss of Hedgehog signalling. It was found that excess of Krz inhibits Hh signalling by promoting Smo internalization and degradation in a clathrin- and proteosomal- dependent manner. Contrary to that observed in vertebrates, the effects of Krz on Smo are independent of Gprk2 activity and of the activation state of the receptor. It is suggested that such differences in Hh signalling are based in the strict requirement of the primary cilia, a structure that is not present in fly epidermal cells, for Hh signalling in most vertebrates (Molnar, 2011).

This work has analysed the requirement of krz during the development of the Drosophila wing disc. The wing disc is an epithelial tissue, and its patterning and growth depends on the activity of several conserved signalling pathways. It was therefore reasoned that any requirement of Krz in the regulation of these pathways should be uncovered by the phenotype of the complete genetic loss of krz in the disc. Surprisingly, it was found that wing discs (and all other imaginal discs) can develop in an almost entirely normal manner in the total absence of Krz function (see also Roman, 2000). This finding implies that any role of Krz during normal development is dispensable for the regulation of the signalling pathways operating in the wing disc. It is emphasized that even small changes in the levels or domains of signalling by the Notch, EGFR and Hh/Smo pathways result in very characteristic and distinct phenotypes in the wing, and consequently it is concluded that these pathways operate normally in the absence of Krz in the discs (Molnar, 2011).

The function of Krz has been linked in imaginal discs with the regulation of Notch protein stability (Mukherjee, 2005) and of MAPK phosphorylation (Tipping, 2010). These conclusions are base on sound biochemical data taken from cell culture experiments, and also on the analysis of genetic interactions evaluating the ability of krz mutations in heterozygosity to modify the phenotypes caused by Notch pathway components and MAPK alleles (Mukherjee, 2005; Tipping, 2010). It was also found that krz reduction enhances the phenotype of a Notch loss-of-function condition, no Notch-related phenotype was found in krz mutant wings. Furthermore, changes were found in Notch accumulation in a small fraction of krz1 and Df(3R)krz mutant clones, in contrast to (Mukherjee, 2005). In this context, it is interesting to note that a robust accumulation of Notch was found when krz mutant cells over-express the Notch ligand Delta, suggesting that the function of krz becomes critical to promote Notch turnover upon Notch-Delta interactions. In this manner, the implication of this analysis and of previous works is that Krz might be required to optimise some aspects of Notch degradation or MAPK phosphorylation, but that these processes can occur normally in the absence of Krz. It might well be that only upon particular alterations of Notch levels, or in sensitized genetic backgrounds, such as over-expressing a non-dephosphorylable form of MAPK, these fine-tuning aspects of Krz are manifested in phenotypic modifications. It is unlikely that the paucity of krz requirements during imaginal development was due to functional redundancy with other arrestin proteins, because the only Drosophila candidate, CG32683, is not expressed in imaginal discs and does not affect imaginal development when over-expressed (Molnar, 2011).

The lack of a krz mutant phenotype in the discs is also surprising considering the multitude of roles assigned to its vertebrate counterparts in the Wnt, IGF, Notch, Smo and TGFβ signalling pathways and in ERK activation promoted by many GPCRs (reviewed in Kovacs, 2009). These roles rely both on the regulation by β-arrestins of receptor internalization and subcellular localization, and also on their functions as scaffold for a variety of proteins involved in cellular signalling. It has to be postulated that insect epithelial cells have evolved arrestin-independent mechanisms to control receptor turnover and signalling, and consequently that arrestin function has become less relevant in these cells. This proposal is compatible with Krz retaining the capability to molecularly interact with similar proteins as its vertebrate counterparts, as Krz possesses both amino- and carboxy-terminal arrestin domains and is 72% similar to the mammalian β-arrestin 2 and 74% similar to β-arrestin 1 (Molnar, 2011).

In contrast to the loss-of-function analysis of krz, the study of its over-expression offers clear-cut indications of its implication in regulating Smo internalization. Thus, over-expression of Krz causes a very specific phenotype of loss-of-Hh signalling, manifested in defects localised in the central part of the wing that in extreme cases lead to the total failure of wing development. These phenotypes are associated to the loss of expression of Hh target genes, confirming that they are caused by reduced Hh signalling. As previously described, increased levels of Krz are extremely effective in reducing Smo accumulation in the cell membrane (Cheng, 2010 and this work). This effect is observed with wild type forms of Smo, with Smo mutated in its phosphorylation sites and with a phospho-mimic Smo protein that is constitutively activated. The elimination of Smo is also observed in posterior cells, indicating that Krz promotes Smo elimination independently of Ptc, and also in anterior cells localised away from the source of Hh, suggesting that Krz affects Smo turnover in the absence of ligand. Finally, the elimination of Smo by excess of Krz is independent of Gprk2 activity, because it is still observed in cells deficient for the Gprk2 gene. Gprk2 is required for the transduction of Smo signal, and when Gprk2 levels are lowered, inactive Smo accumulates at the cell membrane. In the double combination (excess of Krz plus loss of Gprk2), Smo is eliminated, suggesting that Smo unmodified by Gprk2 is still capable to interacting with Krz and being removed. The resulting flies show extreme hh loss-of-function phenotypes, likely the result of both loss of Gprk2-dependent Smo activation and increased, Krz-promoted, Smo turnover (Molnar, 2011).

The ability of Krz to interact with Smo in the Drosophila wing is very specific, since no other alterations in the localization and activity of other receptors, such as Notch or EGFR, (Kovacs, 2009; Ayers, 2010). In addition, β-arrestin 2 promotes, upon GRK phosphorylation, the internalization of activated Smo in human embryonic kidney 293 cells (Chen, 2004). Finally, β-arrestin 2 promotes Smo signalling in zebrafish embryos, and this seems to be a physiological function because it is detected in loss-of-function conditions (Wilbanks, 2004). In contrast, a clear antagonism of Krz on Smo signalling caused by Smo internalization and degradation promoted was observed only by excess of Krz, and this effect of Krz is independent of the Smo phosphorylation state and of Gprk2 activity (Molnar, 2011).

One of the main differences in the Smo signalling pathway between vertebrates and Drosophila is the localization in vertebrates of active Smo to the primary cilium, a structure that is present only in the fly in sensory neurons. It can only be speculated that the necessity to translocate Smo complexes associated with the type II kinesin motor Kif3A to the cilium, a structure not present in fly epidermal cells, imposes a requirement for β-arrestins that is not observed in the fly. Nonetheless, the current results show that the capability of Krz to interact with Smo is retained in Drosophila, and this is revealed upon the over-expression of Krz. Once Krz is bound to Smo it would trigger the formation of clathrin-coated pits that targets Smo for degradation in the proteasome, leading to the insufficiency of Hh signalling that was observed. In this way, it is proposed that Krz has retained some of the molecular targets typical of vertebrate β-arrestins, but that these interactions might not occur at physiological levels of expression, or being redundant with other mechanisms of receptor trafficking and signalling (Molnar, 2011).

TRAF6 is a novel regulator of Notch signaling in Drosophila melanogaster

Notch signaling pathway unravels a fundamental cellular communication system that plays an elemental role in development. It is evident from different studies that the outcome of Notch signaling depends on signal strength, timing, cell type, and cellular context. Since Notch signaling affects a spectrum of cellular activity at various developmental stages by reorganizing itself in more than one way to produce different intensities in the signaling output, it is important to understand the context dependent complexity of Notch signaling and different routes of its regulation. This study identified TRAF6 (Drosophila homolog of mammalian TRAF6) as an interacting partner of Notch intracellular domain (Notch-ICD). TRAF6 genetically interacts with Notch pathway components in trans-heterozygous combinations. Immunocytochemical analysis shows that TRAF6 co-localizes with Notch in Drosophila third instar larval tissues. The genetic interaction data suggests that the loss-of-function of TRAF6 leads to the rescue of previously identified Kurtz-Deltex mediated wing notching phenotype and enhances Notch protein survival. Co-expression of TRAF6 and Deltex results in depletion of Notch in the larval wing discs and down-regulates Notch targets, Wingless and Cut. Taken together, these results suggest that TRAF6 may function as a negative regulator of Notch signaling (Mishra, 2014).

Regulation of Toll signaling and inflammation by β-arrestin and the SUMO protease Ulp1

The Toll signaling pathway has a highly conserved function in innate immunity and is regulated by multiple factors that fine tune its activity. One such factor is β-arrestin Kurtz (Krz), which has been implicated in the inhibition of developmental Toll signaling in the Drosophila melanogaster embryo. Another level of controlling Toll activity and immune system homeostasis is by protein sumoylation. This study has uncovered a link between these two modes of regulation and shows that Krz affects sumoylation via a conserved protein interaction with a SUMO protease, Ulp1. Loss of function of krz or Ulp1 in Drosophila larvae results in a similar inflammatory phenotype, which is manifested as increased lamellocyte production; melanotic mass formation; nuclear accumulation of Toll pathway transcriptional effectors, Dorsal and Dif; and expression of immunity genes, such as Drosomycin. Moreover, mutations in krz and Ulp1 show dosage-sensitive synergistic genetic interactions, suggesting that these two proteins are involved in the same pathway. Using Dorsal sumoylation as a readout, it was found that altering Krz levels can affect the efficiency of SUMO deconjugation mediated by Ulp1. These results demonstrate that β-arrestin controls Toll signaling and systemic inflammation at the level of sumoylation (Anjum, 2013).

Hedgehog-regulated ubiquitination controls smoothened trafficking and cell surface expression in Drosophila

Hedgehog transduces signal by promoting cell surface expression of the seven-transmembrane protein Smoothened (Smo) in Drosophila, but the underlying mechanism remains unknown. This study demonstrates that Smo is downregulated by ubiquitin-mediated endocytosis and degradation, and that Hh increases Smo cell surface expression by inhibiting its ubiquitination. Smo is ubiquitinated at multiple Lysine residues including those in its autoinhibitory domain (SAID), leading to endocytosis and degradation of Smo by both lysosome- and proteasome-dependent mechanisms. Hh inhibits Smo ubiquitination via PKA/CK1-mediated phosphorylation of SAID, leading to Smo cell surface accumulation. Inactivation of the ubiquitin activating enzyme Uba1 or perturbation of multiple components of the endocytic machinery leads to Smo accumulation and Hh pathway activation. In addition, this study found that the non-visual beta-arrestin Kurtz (Krz) interacts with Smo and acts in parallel with ubiquitination to downregulate Smo. Finally, it was shown that Smo ubiquitination is counteracted by the deubiquitinating enzyme UBPY/USP8. Gain and loss of UBPY lead to reciprocal changes in Smo cell surface expression. Taken together, these results suggest that ubiquitination plays a key role in the downregulation of Smo to keep Hh pathway activity off in the absence of the ligand, and that Hh-induced phosphorylation promotes Smo cell surface accumulation by inhibiting its ubiquitination, which contributes to Hh pathway activation (Li, 2012).

beta-arrestin Kurtz inhibits MAPK and Toll signalling in Drosophila development

β-Arrestins have been implicated in the regulation of multiple signalling pathways. However, their role in organism development is not well understood. This study reports a new in vivo function of the Drosophila β-arrestin Kurtz (Krz) in the regulation of two distinct developmental signalling modules: MAPK ERK and NF-κB, which transmit signals from the activated receptor tyrosine kinases (RTKs) and the Toll receptor, respectively. Analysis of the expression of effectors and target genes of Toll and the RTK Torso in krz maternal mutants reveals that Krz limits the activity of both pathways in the early embryo. Protein interaction studies suggest a previously uncharacterized mechanism for ERK inhibition: Krz can directly bind and sequester an inactive form of ERK, thus preventing its activation by the upstream kinase, MEK. A simultaneous dysregulation of different signalling systems in krz mutants results in an abnormal patterning of the embryo and severe developmental defects. These findings uncover a new in vivo function of β-arrestins and present a new mechanism of ERK inhibition by the Drosophila β-arrestin Krz (Tipping, 2010).

This study demonstrate that the Krz protein is necessary for setting a precise level of activation of two maternal signalling pathways, Torso and Toll. This activity of Krz helps to establish the correct domains of expression of developmental patterning regulators that are under the control of these pathways (Tipping, 2010).

Genetic and protein interaction data suggest a new mechanism by which Krz may limit the activity of Torso. It was observed that Krz preferentially binds and sequesters an inactive form of ERK, thereby making it unavailable for activation by the upstream kinases such as MEK. Such a mechanism of direct inhibition of ERK activation by β-arrestin binding has not been previously reported. This mechanism is consistent with the observed in vivo effects of loss of krz on ERK activity. In krz maternal mutant embryos, ERK is not sequestered and therefore more ERK is available to transduce Torso signals, resulting in hyperactivation of Torso target genes, tll and hkb. Furthermore, consistent with this model is the observation that Krz and MEK apparently compete for ERK when all three proteins are co-expressed in S2 cells (Tipping, 2010).

Interaction assays using mutated forms of Krz and ERK indicate that the conformations of both proteins have an effect on their binding affinity. On binding to an activated GPCR, the arrestin molecule undergoes a dramatic conformational change that can be mimicked by specific mutations (Gurevich, 2004). In immunoprecipitation experiments it was observed that such 'pre-activated' form of Krz (R209E) has a much greater affinity for ERK, compared with the wild-type Krz protein, and that this higher affinity is also observed for the equivalent mutant of human β-arrestin2. This suggests that the ERK-binding ability of β-arrestin may be affected by its conformation, but it is unknown at present whether any upstream signals convert Krz into an activated form in the embryo. Overexpression of Krz-R209E using the da-GAL4 driver did not result in any observable phenotype and could rescue zygotic loss of krz, suggesting that it retains most of the functions of wild-type Krz (data not shown) (Tipping, 2010).

It was observed that the conformation of ERK itself has a large effect on its interactions with Krz. In the binding experiments, activated forms of ERK bind Krz (and human β-arrestin2) with lower affinity, compared with wild-type inactive ERK. Moreover, mutations in the TEY motif, which render ERK constitutively inactive, also lower its affinity for Krz, which is at a first glance a surprising result. However, previous studies have shown that both types of mutations in the TEY motif, which is a part of the activation loop, increase disorder in the lip region and cause a conformational change in the ERK molecule that makes it different from the basal state. It is therefore speculated that the activation loop may be involved in mediating an interaction of ERK with β-arrestin. Consistent with the current results, deviation of ERK structure from the basal state would decrease its association with β-arrestin (Tipping, 2010).

Other studies have reported formation of protein complexes containing β-arrestins and an activated form of ERK. It is possible that in those experimental conditions other binding partners, such as Raf or the activated receptor, assist in stabilizing the complex of MAP kinases with β-arrestin. This study has shown that although Krz can bind to the Drosophila homologues of both MEK and Raf, overexpression of Krz does not increase production of dpERK by the MAPK cascade downstream of activated RTKs, but instead appreciably inhibits it in the absence of overexpressed Raf. The data do not rule out a possibility that Krz may still promote ERK activation in other biological contexts, particularly downstream of activated GPCRs, but this question awaits further investigation (Tipping, 2010).

Interestingly, the sequestration mechanism of ERK inhibition described in this study is different from the effects of Krz on Notch. Previous studies have shown that Krz inhibits Notch activity by forming a ternary complex with Deltex and the Notch receptor. Formation of this complex increases Notch turnover and thereby downregulates Notch signalling (Mukherjee, 2005). No change was observed in ERK turnover in the presence of wild-type overexpressed Krz, suggesting that Krz is unlikely to be involved in the regulation of ERK stability. However, given the versatility of molecular functions displayed by β-arrestins, it is possible that there are other, as yet uncharacterized mechanisms by which Krz controls signalling downstream of RTKs (Tipping, 2010).

The inhibitory effects of Krz on ERK activity are not limited to the Torso pathway and early embryogenesis, but are also observed in other tissues and at later developmental stages. Thus, broadening of the dpERK patterns activated by EGFR and Btl was observed in krz maternal mutant embryos. An increase in the overall levels of dpERK during mid-to-late embryogenesis was also detected on western blots. Later in development, ERK is activated by EGFR in the wing and both EGFR and Sevenless in the eye. Genetic data suggest that Krz also inhibits ERK activity in these tissues during larval development. A broad involvement of Krz in inhibiting ERK activity suggests that Krz has a general inhibitory role to limit the activity of different RTKs in Drosophila development (Tipping, 2010).

In addition to its effects on RTK signalling, it was observed that Krz has an important role in limiting the activity of the Toll receptor, which specifies the development of the ventral structures. Other studies have reported that mammalian β-arrestins can downregulate NF-κB signalling by binding and stabilizing the NF-κB inhibitor IκBα. The inhibitory effects of Krz on Dorsal may involve a similar mechanism. It was observed that Krz can directly bind to the Drosophila orthologue of IκBα, Cactus, suggesting that the mechanism of NF-κB inhibition by β-arrestins at the level of IκBα may be conserved. Consistent with this finding, a decrease was detected in the level of the Cactus protein in krz maternal mutants at 0-4 h of development, which may explain the observed expansion of the nuclear gradient of Dorsal in these mutants. It is still unclear why expansion of Dorsal nuclear localization is more pronounced in the posterior half of the embryo (Tipping, 2010).

In the developing embryo, the Torso and Toll pathways do not work in isolation, but are involved in cross-regulatory interactions on certain common targets, such as zen. zen is repressed by nuclear Dorsal in the ventral part of the embryo, and relieved of this repression (de-repressed) by the signalling activity of Torso emanating from the embryo poles. The molecular mechanism of this de-repression is still unknown. It was observed that loss of krz shifts the balance of the effects of Torso on Toll, which results in an inappropriate expansion of zen expression at the embryo poles. It is speculated that Krz helps Torso to achieve a precise level of de-repression of zen by limiting the activity of ERK. Krz is thus able to control the separate activities of the Torso and Toll pathways (reflected in its effects on tll, hkb, twi, and rho), as well as regulate common Torso and Toll targets such as zen. For such pathways that are engaged in cross-regulatory interactions, Krz ensures that a proper level of signalling activity from one pathway reaches the other. This function adds an important new mechanism to understanding of the ways in which signalling pathways are coordinately regulated during development (Tipping, 2010).

A ubiquitous distribution of Krz in the embryo agrees with the dysregulation of multiple pathways observed in krz mutant animals. As overexpression of Krz does not cause any obvious defects, the level of Krz itself is not limiting for the regulation of signalling. Instead, Krz apparently makes other signalling co-factors limiting for their respective pathways, essentially working as a molecular 'sponge' to prevent pathway hyperactivity. Specificity of Krz function is likely to be determined by its selective interactions with specific pathway co-factors. Maternal loss of krz function thus affects multiple developmental signalling pathways, resulting in an accumulation of defects that ultimately lead to severe morphological abnormalities such as a disruption of gastrulation movements. By analysing the effects of loss of krz on individual pathways in vivo, this study has been able to show its role in the regulation of RTK and Toll signalling. Future studies will likely reveal other pathways and levels of regulation that are under the control of the Drosophila β-arrestin Krz (Tipping, 2010).

Functional characterization of kurtz, a Drosophila non-visual arrestin, reveals conservation of GPCR desensitization mechanisms

The arrestins are a family of molecules that terminate signaling from many different G protein-coupled receptors, by inhibiting the association between receptor and downstream effectors. a human βarrestin2-GFP fusion protein has been used to explore the dynamics of different neuropeptide receptors in Drosophila and a βarrestin translocation assay has been used to identify ligands at orphan receptors. This study reports that the Drosophila arrestin encoded by kurtz functions in a similar fashion and can be employed to investigate GPCR-arrestin associations. Specifically, a GFP-krz fusion protein, upon co-expression with various Drosophila peptide receptors, an amine receptor, and a mammalian peptide receptor translocates to the plasma membrane in specific response to ligand application. This molecular phenotype is exhibited in a mammalian cell line as well as in a Drosophila cell line. Notably, the details of receptor-arrestin associations in terms of endocytotic patterns are functionally conserved between the mammalian arrestins and Kurtz. Furthermore, kurtz mutants exhibit hypersensitivity to osmotic stress, implicating GPCR desensitization as an important feature of the endocrine events that shape this stress response (Johnson, 2008).

Exploratory activity in Drosophila requires the kurtz nonvisual arrestin

When Drosophila adults are placed into an open field arena, they initially exhibit an elevated level of activity followed by a reduced stable level of spontaneous activity. The initial elevated component arises from the fly's interaction with the novel arena since: (1) the increased activity is independent of handling prior to placement within the arena, (2) the fly's elevated activity is proportional to the size of the arena, and (3) the decay in activity to spontaneous levels requires both visual and olfactory input. These data indicate that active exploration is the major component of elevated initial activity. There is a specific requirement for the kurtz nonvisual arrestin in the nervous system for both the exploration stimulated by the novel arena and the mechanically stimulated activity. kurtz is not required for spontaneous activity; kurtz mutants display normal levels of spontaneous activity and average the same velocities as wild-type controls. Inhibition of dopamine signaling has no effect on the elevated initial activity phase in either wild-type or krz1 mutants. Therefore, the exploratory phase of open field activity requires kurtz in the nervous system, but is independent of dopamine's stimulation of activity (Liu, 2007).

An understanding of the significance of the elevated initial activity brought about by the open field arena requires better insight into the relevance of this behavior for the fly. When trying to decipher the ethological basis of a specific behavior, one should consider the proximal causes of a behavior, including the inducing stimuli and the neurobiological requirements for the expression of that behavior, an evolutionary relationship for the behavior, and whether the behavior has any survival value. In dissecting the elevated initial activity, it has been shown that the magnitude of the effect is dependent on the properties of the arena, independent of experimenter handling, and that the cessation of the elevated activity is dependent on vision and, to a lesser extent, on olfaction. It has also been shown that elevated initial activity is present in diverged species, suggesting that it may underlie an important and general survival function. On the basis of these observations, it is proposed that the elevated initial activity constitutes exploration. This study refers to the following definition of exploration: exploration is evoked by novel stimuli and consists of behavioral acts and postures that permit the collection of information about new objects and unfamiliar parts of the environment. A specific defect in exploration may arise either from a failure to detect or process the novel stimuli or from a failure in motivation and executive function (Liu, 2007).

This hypothesis of Drosophila exploration occurring during the elevated initial activity phase in a circular open field arena is supported by the demonstration that the proximal causes of this stimulated activity come from properties of the arena and not from handling. The amount of activity during the first minute in a circular arena responded significantly to changes in arena size, indicating that some property of the arena itself is a proximal cause for this behavior: the greater the area to explore, the greater the amount of activity required to habituate the novelty stimulus. Sensory-deprived flies are deficient in the decay from elevated initial activity, which also supports the exploration hypothesis. The failure to visually observe the surroundings leads to an inability to recognize the surroundings and habituate to the stimulus, prolonging the exploration. The decline in exploration was also inhibited in the anosmic or83b2 mutants, suggesting that olfaction also has a role in the habituation to the novelty of the arena. The absence of an activity deficit in the antenna-transformed AntpNS/+ flies suggests that this sensory organ is largely dispensable for activation of exploration. The gross structural changes in AntpNS/+ flies lead to severe defects in olfactory jump responses. Hence, it is apparent from AntpNS/+ that the elevated initial activity found in the open field arena and the olfactory jump responses have different sensory requirements (Liu, 2007).

Previous evidence has suggested that dopamine may generally regulate stimulated activity, including: initial activity within an open field, ethanol-vapor-stimulated activity, and mechanically stimulated activity. The only other mutation known in Drosophila to specifically affect initial activity in an open field arena activity is a spontaneous allele of the tyrosinase-1 gene; the tyr11 allele results in flies with significantly higher levels of initial activity than wild-type flies, but no differences are found in the level of spontaneous activity. The tyr11 allele was identified as a spontaneous mutation having only 70% of the normal levels of dopamine, the molecular identity of this mutation.Dopaminergic signaling has been selectively inhibited with the targeted expression of tetanus toxin light chain in the majority of tyrosine-hydroxylase-expressing cells. The resulting flies were hyperactive after being banged to the bottom of a cylinder in a negative geotaxis assay but apparently have normal locomotion in the absence of this mechanical stimulation. In another approach, dopamine synthesis was inhibited with 3-IY, an inhibitor of tyrosine hydroxylase that reduces total dopamine levels to ∼10% of the wild-type levels. The 3-IY-treated flies initially displayed normal activity in a 6-cm2 arena; however, after stimulation with ethanol vapor, the activity of the 3-IY-treated flies was reduced relative to the untreated flies. These studies suggest that dopamine has a role in stimulated activity, although the direction of the response may differ, depending on either the treatment or the stimulus. There are, however, clear differences in how these approaches disrupt the dopamine pathway. In the transgenic approach, most but not all dopaminergic neurons express the toxin, leaving a small number of dopamine-signaling pathways intact. In both the tyr11 mutation and the pharmacologic inhibition of dopamine synthesis, it is not known if synthesis is globally inhibited. It also remains possible that the residual dopamine levels in these flies may have enhanced effects due to a sensitization of their respective circuits (Liu, 2007).

More recently, several studies have highlighted a role for dopamine in regulating spontaneous activity and arousal in Drosophila. It has been found that activation of dopaminergic pathways results in state-dependent locomotor responses. In most flies, photostimulated dopamine release leads to an immediate increase in locomotor activity; however, in flies already expressing higher levels of ambulation, the forced release of dopamine leads to lower levels of activity. In two studies where synaptic levels of dopamine were likely to be increased, significant increases in locomotor activity were found. The inhibition of dopamine reuptake with cocaine activates locomotor activity, whereas very high doses of this drug suppress ambulation. A mutation has been identified in a dopamine transporter, fumin, that results in dramatically increased levels of spontaneous activity. The absence of fumin in neurons and glia may lead to higher synaptic levels of dopamine. These recent studies signify that dopamine probably has a dual role in regulating activity: an increase in locomotor activity occurs with lower levels of dopamine and higher levels suppress spontaneous activity (Liu, 2007).

It is possible that krz regulates elevated initial activity through an effect on dopamine signaling. If one presupposes that, in the developmentally rescued krz1 homozygotes, G-protein-coupled receptor signaling is generally extended and amplified due to the absence of agonist-dependent desensitization, then the reduction in the exploratory activity phase may result from an increased sensitivity to the effects of dopamine on activity. However, the data failed to show any effect of inhibition of dopamine signaling on the exploratory activity phase of wild-type flies or the developmentally rescued krz1 homozygotes, indicating that dopamine is not required in flies for the exploratory activity phase in an open field arena (Liu, 2007).

Spontaneous activity was dramatically reduced after feeding wild-type flies 3-IY, consistent with the increase in activity found with photostimulated dopamine release, with the increase in synaptic dopamine levels in the fumin mutant, or after cocaine treatment. The affect of 3-IY on spontaneous activity was not found in the krz1 homozygous flies. Since the krz1 mutation suppresses the effect of 3-IY on spontaneous activity, krz most likely acts as a negative regulator of dopamine signaling during spontaneous activity. In the absence of krz, the dopamine receptors may perdure in an activated state, compensating for the shortfall of dopamine in the 3-IY-treated flies (Liu, 2007).

A considerable conservation was found of the open field behavior in three species of drosophilids. D. simulans and D. melanogaster are closely related members of the melanogaster species group, having diverged ∼2.5-4 million years ago (MYA). In contrast, the last shared ancestor for D. virilis and D. melanogaster is thought to have lived ∼65-70 MYA during the late Cretaceous period. Remarkably, however, a potentially homologous behavior is also present in many species of vertebrates. The presence of a conserved behavioral response in such divergent species strongly suggests that it provides general advantages, for example, leading the animal to new food sources, mating partners, or protective shelter (Liu, 2007).

The responses to an open field arena have been most thoroughly studied in rodents where the behavior in an open field arena is thought to be shaped by at least two conflicting internal drives: emotionality and curiosity. The emotionality factor is thought to represent anxiety or fear and is typically measured by an inhibition of ambulation, an increased number of defecations, and decreased entries into the center of the open field. These three ethological parameters in the open field arena are frequently reversible with anxiolytic drugs and enhanced by anxiogenic drugs, strengthening the association of these behaviors with anxiety. However, factor analysis has shown that anxiety in rodents is multidimensional, with decreases in locomotion and defecation influenced by different factors. In rodents, the inhibition of ambulation in the open field arena by an anxiety-like factor appears to be initially countered by a curiosity drive that leads to increased exploration (Liu, 2007).

Outwardly, the behavior of the drosophilids in the open field resembles that of rats and mice: they all have an initially elevated period of activity, avoid the center of the arena, and prefer corners. It is not clear whether these behaviors of the drosophilids are functional analogs to the rodent behaviors since there is as yet much less known about the motivational factors that drive Drosophila behavior. The stressors of hyperagitation prior to placement in the arena or of social isolation do not affect the Drosophila exploratory activity phase, indicating that, if an emotionality factor exists in Drosophila, these potential stressors are insufficient to affect exploration. It is expected, however, that an exploratory drive similar to the one in rodents may exist in flies since it would serve the important survival functions of finding food, mating partners, or shelter. In fact, one explanation of the strong preference for corners and the striking thigmotactic responses in Drosophila is that the flies are seeking shelter. An increase in the size of a square arena resulted in a decrease in the amount of activity during the first minute -- the opposite of the result in circular arenas. Although the corners inhibited the spontaneous activity phase independently of arena size, the exploration phase was most inhibited when the corners were farther apart: as the distance between shelters increases, the propensity to explore decreases. The phenomenon of staying in sheltered areas may also be associated with the avoidance of the arena's center and may represent the expression of fear or anxiety in Drosophila (Liu, 2007).

Alternatively, the centrophobicity and corner preference may be independent of any anxiety-like construct. Drosophila may have a powerful innate thigmotactic response that is independent of shelter seeking. In this case, the corners would offer more surface to rub against and would therefore become the preferred location. This later explanation is not favored since the flies are, in general, not continuously rubbing against the arena's edge as much as maintaining proximity to it. Drosophila may also have innate search strategies that drive them to the arena's edge through biased orientation and a persistence of direction during walking (Liu, 2007).

The rescue of the krz1 elevated initial activity deficit with both a genomic transgene and the pan-neural expression of a krz cDNA demonstrates a requirement for this gene in the nervous system for the expression of this activity phase. Since the krz1 homozygotes can ambulate at speeds identical to those of wild-type flies during the first minute within the arena and have wild-type levels of spontaneous activity in either the open field or the Trikinetics activity monitor, the krz1 phenotype in exploration is not due to a defect in motor function. Consequently, the requirement for krz in exploration more likely lies in either sensory- or executive-function-level processing. The developmentally rescued krz1 homozygotes have reduced olfactory sensitivity and antennal structural defects, which exhibit variable penetrance and expressivity (Ge, 2006). However, the olfaction defect appears not to be the proximal cause of the activity deficit, since the olfaction phenotype can be rescued without altering the exploration phenotype, and olfaction is dispensable for the exploration phase. The selection of krz1 homozygotes with or without antenna defects failed to affect the exploratory activity phase, suggesting that this exploration phenotype is independent of the antennal defect. Moreover, the more severe structure defects in AntpNS/+ flies failed to produce an elevated initial activity phenotype, further suggesting that the antennal structural defect is not responsible for the exploration phenotype seen in the developmentally rescued krz1 homozygotes. As the rescued krz1 homozygotes are unresponsive to novelty and mechanical agitation, this gene may have a more central role in gating the responses to certain forms of stimulation (Liu, 2007).

In the large square arena, the krz1 homozygotes behave indistinguishably from wild type: the same preference for corners, the same avoidance of the center of the arena, and the same activity profiles. In contrast, the rescued krz1 homozygotes show an exploration deficit in the small square arena, where the corners do not seem to inhibit exploration to nearly the same extent as in the larger arena. The presence of an initial activity deficit in both the circular arena and the smaller square arena and the absence of the initial activity deficit in large square arenas would occur if either (1) the repression of activity in large square arenas indirectly masks the krz1 deficit or (2) it more directly compensates for the absence of krz. An example of the former would be if the preference for corners is a general inhibitor of all locomotion, reducing the exploratory activity phase to a floor level commensurate with that of the developmentally rescued krz1 homozygotes. In an example of the later case, the effect of the corners may specifically reduce exploration by satiating a portion of the drive, which is reduced in krz1 homozygotes. In either case, the results do not argue against krz activity being required for exploration. The absence of this krz exploration phenotype also illustrates that tests of activity in some square arenas are restrictive—not all parameters of activity are identifiable in the 22.4-cm2 arena (Liu, 2007).

Mice have two nonvisual arrestins, βarr1 and βarr2 (Conner, 1997; Bohn, 1999). These proteins are required for the agonist-dependent desensitization of GPCRs and for the regulation of a number of other cell-surface molecules (Lefkowitz, 2004). The mouse βarr2−/− mutation also results in reduced locomotor activity, although it appears to affect both the elevated initial activity and the plateau phases of activity (Bohn, 2003). The causes of the innate βarr2−/− defect in activity are not currently known; these mice may have problems with emotionality or perhaps they have minor motor defects. The murine βarr2 may also have different roles from those of krz in Drosophila in regulating locomotion; however, there are also significant experimental differences in how these experiments were performed. The βarr2 mutants developed without this gene's activity, whereas the krz mutant flies were periodically supplied with krz activity throughout development (Bohn, 2003). The two nonvisual arrestins in mice are also at least partially redundant, whereas krz is the only nonvisual arrestin in Drosophila (Liu, 2007).

In humans, subtle defects in the agonist-dependent desensitization of G-protein-coupled receptors may be a considerable contributing factor to the severity of affective disorders. A variant of the human G-protein-coupled receptor kinase 3 gene has been identified as a candidate locus for bipolar disorder. The levels of GRK3 are lower in the leukocytes of patients with severe bipolar symptoms. In the postmortem brains of depressed patients, there is a significant increase in the level of membrane-bound GRK2, which is not found in the brains of antidepressant-treated patients; βARR2 may be coordinately regulated with GRK2 in the brains of these depressed patients. A strong correlation has been found between lower βARR1 levels in leukocytes and the severity of patients with major depression. A greater understanding of arrestin function within the nervous system is required to understand how agonist-dependent desensitization of protein-coupled receptors may lead to pathological emotional states. Although these states are probably controlled by different neurotransmitter systems in Drosophila, they still most likely involve G protein signaling. Drosophila, with a single nonvisual arrestin and a simpler behavioral repertoire, provides an excellent opportunity for examining the role of agonist-dependent desensitization in behavior. Understanding how krz is involved in the responses to novelty and mechanical stimulation will likely provide important insights into how these molecules regulate emotional responses in vertebrates (Liu, 2007).

A Drosophila nonvisual arrestin is required for the maintenance of olfactory sensitivity

Nonvisual arrestins are a family of multifunctional adaptor molecules that regulate the activities of diverse families of receptors including G protein-coupled receptors, Frizzled, and Transforming growth factor-β receptors. These activities indicate broad roles in both physiology and development for nonvisual arrestins. Drosophila has a single nonvisual arrestin, kurtz, which is found at high levels within the adult olfactory receptor neurons (ORNs), suggesting a role for this gene in modulating olfactory sensitivity. Using heat-induced expression of a krz cDNA through development, krz1 lethality was rescued. The resulting adults lacked detectable levels of krz in the olfactory system. The rescued krz1 homozygotes have an incompletely penetrant antennal structural defect that was completely rescued by the neural expression of a krz cDNA. The krz1 loss-of-function adults without visible antennal defects displayed diminished behavioral responsiveness to both aversive and attractive odors and also demonstrated reduced olfactory receptor potentials. Both the behavioral and electrophysiological phenotypes were rescued by the targeted expression of the krz cDNA within postdevelopmental ORNs. Thus, krz is required within the nervous system for antennal development and is required later in the ORNs for the maintenance of olfactory sensitivity in Drosophila. The reduced receptor potentials in krz1 antenna indicate that nonvisual arrestins are required for the early odor-induced signaling events within the ORNs (Ge, 2006).

This study has demonstrated that there are at least two requirements in olfaction for the krz nonvisual arrestin: one developmental and the other postdevelopmental. The adult rescued loss-of-function krz mutations have an incompletely penetrant structural defect in the antenna. This recessive phenotype is rescued by the expression of a krz cDNA in the nervous system but not by the late expression of krz within the ORNs. The krz1 mutants without the visible structural defects have a blunted behavioral responsiveness to odorants. This latter phenotype is a general shift toward reduced sensitivity at several concentrations and affects both aversive and attractive odorants. The defect in olfactory sensitivity was shown to be recessive and was mapped to the krz locus both by a genomic transgene and through the targeted expression of a krz cDNA within the ORNs. The behavioral defect is also accompanied by a reduced odor-induced receptor potential within the third antennal segment. The reduced EAG amplitude was also recessive and can be rescued by late expression of the krz cDNA within the ORNs. Thus, krz -- the only nonvisual arrestin in Drosophila -- is required essentially postdevelopmentally for generating normal receptor potentials after olfactory stimulation, and this reduced responsiveness to odors may account for most of the reduced sensitivity in olfactory behavioral responses (Ge, 2006).

An important question arising from this study is when krz is required for these two requirements. The krz gene is broadly expressed in the late third instar antennal imaginal discs, consistent with a role for this gene during development of this organ (Roman, 2000). The variable penetrance and expressivity of the structural antennal defect in the rescued krz1 homozygotes are most likely due to differences in the induced expression of krz between individuals during development, which may relate to how old they are when krz induction ceases. The induction of krz activity in wandering third instar homozygotes results in a very low rate of antennal defects (Roman, 2000; this study). However, in the current preparation of krz mutants, some homozygotes will receive the final heat shock induction of krz activity as a late L2 to an early L3 instar and, as a consequence, may have less krz gene product available during early to mid metamorphosis, when the antenna structure develops, than the older flies. This hypothesis is also consistent with THE observation that the later a fly ecloses, the more severe is the antennal phenotype. During metamorphosis, most of the rescued krz homozygotes die shortly after the heat shock and none will eclose (Roman, 2000). Therefore, a more thorough dissection of the timing of this krz requirement is difficult. Nevertheless, since the pan-neuronal (c155)-rescued krz homozygotes have completely normal antenna, the foci for this developmental requirement is neuronal. The activation of krz activity in the ORN with the OR83bGal4 drivers is too late to rescue the developmental defect. It is also not clear whether the ORNs are responsible for the krz-defined developmental requirement (Ge, 2006).

The krz gene product is present in adult ORNs, suggesting a continued physiological requirement for this gene. The rescue of both the olfactory avoidance and the reduced receptor potential phenotypes with the OR83bGal4 driver places the earliest time point for a krz requirement for these responses at just before eclosion. By this time (~85 h APF), the ORNs have all differentiated, the antennal nerve has stopped expanding, ORN axons have found their targets in the antennal lobe, and at least some of the ORs have begun to accumulate in the cell bodies awaiting transfer to the cilia. The OR83b gene is required to shuttle most ORs into the cilia, allowing for proper subcellular expression of these receptors. The krz gene product may function in the maturation of the dendritic component of the ORN, and it may have a function that would be persistently required for odor sensitivity. However, since the krz1 mutants can respond to odors, this gene is not essential for the odor response, indicating that either there are redundant functions for this gene or krz acts as a modifier of the olfaction-signaling pathway (Ge, 2006).

The earliest step in odor-induced signal transduction within the Drosophila ORNs is thought to involve the activation of the ORs within the cilia membranes. These ORs are members of a GPCR family, although it is not known if they, in fact, couple to G proteins. In Xenopus oocytes, however, the OR43a protein was capable of coupling with a human Gα 15-containing G protein, suggesting that these receptors may also couple to heterotrimeric G proteins in vivo. The vertebrate nonvisual arrestins regulate the activity of GPCRs in two ways: first, by binding to phosphorylated, agonist-bound receptors and thereby inhibiting further interactions of the activated GPCR with heterotrimeric G proteins and, second, by drawing the receptor into the endocyotic machinery where the GPCR is dephosphorylated and can be recycled back to the plasma membrane. Nonvisual arrestins promote the endocytosis of activated GPCRs through the clathrin-binding domain and an adaptor protein (AP)-2-binding domain. The interaction of nonvisual arrestins with the β 2-adaptin subunit of the AP-2 appears to be the rate-limiting step in bringing the GPCR-arrestin complex into clathrin-coated pits (Kirchhausen, 1997). Most nonvisual arrestin-GPCR complexes dissociate at the coated pit, but in a few cases, the complex persists through internalization (Pitcher, 1995; Krueger, 1997; Oakley, 1999). The activity of nonvisual arrestins is not limited, however, to members of the GPCR superfamily but includes the ability to bind to and internalize a larger group of membrane receptors (Lefkowitz, 2005; Ge, 2006 and references therein).

Since the primary step in olfactory signal transduction is the activation of ORs within the cilia of the ORNs, an evident hypothesis is that nonvisual arrestins have the specific role of regulating the responsiveness of these neurons through agonist-dependent desensitization of these receptors. In rat olfactory membranes, pretreatment with neutralizing antibodies against GRK3 or β arrestin-2 lead to dramatic increases in odor-induced cAMP production, which indicates that the GRK3 and β arrestin-2 are most probably desensitizing the OR within these sensory neurons. In contrast, the loss of GRK activity in mutants results in a diminished sensitivity to odorants. The deletion of the GRK3 gene results in mice that have reduced odor-induced cAMP production and hence most probably reduced olfactory sensitivity. Mutants of the grk-2 gene in C. elegans also have decreased behavioral and physiological responses to odorants. The reduced sensitivity to odorants in these mutants would suggest that the chronic loss of GRK activity leads to a compensatory mechanism that downregulates early events in olfactory signal transduction. It is not presently clear what this mechanism may be. The phenotypes of the mouse and C. elegans GRKs are analogous to those that reported in this study for the krz gene of Drosophila. It is interesting that loss-of-function mutants in the only arrestin in C. elegans displaying wild-type behavioral sensitivity to odorants, suggesting that the grk-2 gene product can regulate ORs in an arrestin-independent manner (Fukuto, 2004; Palmitessa, 2005; Ge, 2006 and references therein).

Visual arrestins differ from nonvisual arrestins both in their patterns of expression and structurally (Craft, 1995). The vertebrate visual arrestins are specialists; they are located almost exclusively in photoreceptor cells and function primarily to desensitize the opsins. The vertebrate β Arr1 and β Arr2 nonvisual arrestins are generalists; they are expressed in most tissues and can desensitize a large number of GPCRs and non-GPCRs (Parruti, 1993; Sterne-Marr, 1993; Gurevich, 1995; Lefkowtiz, 2005). The vertebrate and Drosophila visual arrestins do not have obvious clathrin-binding domains or AP-2-binding domains similar to those located near the carboxyl end of the non-visual arrestins. These two arrestin classes are similar in how they bind to activated GPCRs but differ in the events subsequent to binding. The visual arrestins are released from rhodopsin after this receptor is dephosphorylated on the disc membrane of the rod outer segment (Ge, 2006).

In Drosophila, there are two visual arrestins: arr1 and arr2. A third Drosophila gene, CG32683, is identified as an arrestin by significant sequence similarity in both the amino- and carboxy-terminal domains, though it is about twice the size of the other arrestins; this atypical arrestin also lacks both the clathrin-binding and AP-2 adaptor-binding domains that structurally and functionally differentiate the nonvisual arrestins from the visual arrestins, suggesting that it may operate as a third visual arrestin. The arr1 and arr2 genes are expressed in the antenna and maxillary palps in addition to the photoreceptor neurons, while the expression of CG32683 is presently uncharacterized. Loss-of-function mutations in either of the two characterized visual arrestins have variably reduced EAG amplitudes. The individual arr1 and arr2 mutants displayed different electrophysiological responses to different odors and odor concentrations; these responses were frequently more severe in the arr1, arr2 double mutant, suggesting a functional redundancy between the two genes. Additionally, these mutants had minor differences in olfactory adaptation as measured by EAGs. Together, these results indicate a role for these two visual arrestins in modulating olfactory responsiveness (Ge, 2006 and references therein).

Although the data demonstrate a role for krz in regulating the early events in olfactory signal transduction, it is not clear whether krz affects ORs through the canonical desensitization-resensitization cycle of vertebrate nonvisual arrestins. Even though KRZ is found, albeit at low levels, within the sensilla and therefore may directly interact with the activated ORs, it was not found that krz had a considerable effect on olfactory adaptation to octanol (OCT) and methylcyclohexanol (MCH}; the kinetics of desensitization and resensitization of the receptor potentials in the krz1 homozygotes were similar to wild-type controls. The reduced agonist-dependent desensitization of ORs predicted in krz1 homozygotes would be expected to lead to a prolonged activation of downstream effectors. The resulting increase in secondary messengers should lead to increased adaptation. The absence of a prominent and general effect on olfactory adaptation in the krz1 homozygotes could be reconciled with the krz-dependent desensitization of ORs model either if there is a functional redundancy with the visual arrestins or if a separate compensating mechanism may mask this effect. The visual arrestins are also expressed in the ORNs and may provide functional redundancy. Moreover, the differences between the effects of short-term inhibition of GRK3 and the GRK3 mutations suggest the presence of such a compensating mechanism in the rodent ORNs, which may also exist in the Drosophila ORNS. It also remains possible that krz is not required for the agonist-dependent desensitization of ORs but has a different role in regulating early events in olfactory signal transduction (Ge, 2006).

It is not known if Drosophila ORs are internalized after odor presentation. In catfish, agonist-bound metabotropic glutamate receptors are internalized into the dendrites and cell bodies of ORNs through a clathrin- and dynamin-dependent pathway, indicating that the machinery for internalization of ORs is present in these cells. Nevertheless, the internalization and resensitization of desensitized ORs seems unlikely to occur within the olfactory cilia through the clathrin-dependent internalization pathway. The Drosophila olfactory cilia are long, branched processes that are as thin as 50 nm in diameter within the large basiconic sensilla (Shanbhag, 1999). Clathrin-coated vesicles appear to come in two sizes: 60 or 100 nm in diameter. Thus, even if a single clathrin-coated vesicle was internalized within a basiconic cilium, it would likely occlude protein trafficking and perhaps inhibit signaling from distal parts of the dendrite. It seems unlikely therefore that arrestin-dependent internalization of ORs would take place within the cilia. In both frog photoreceptor neurons and in the olfactory neurons of C. elegans, specialized areas of clathrin-mediated vesicle sorting are located at the base of the rod outer segment and olfactory cilia, respectively. A similar region of specialization may exist in Drosophila ORNs, where coated vesicles have been found forming at the base of the olfactory cilia. It will be interesting to see if KRZ is involved in the removal of ORs from the cilia membrane or the recycling of ORs back to the cilia membranes at these sites and whether this function can account for the reduced olfactory sensitivity found in the krz1 mutants (Ge, 2006).

Understanding how organisms perceive and respond to the environment is a fundamental problem for behavioral neuroscience. GPCRs are sentries for much of the information that an organism perceives. A better understanding is needed of the mechanisms that regulate the fidelity of these sensory receptors before how organisms react to and interact with external stimuli can be fully understood. Nonvisual arrestins are among the most important regulators of the activities of GPCRs. This study has shown that a mutant of a nonvisual arrestin has reduced, but not eliminated, olfactory sensitivity. Thus, this molecule is required for the preservation of the full range of olfactory responsiveness in Drosophila. The further characterization of the role of krz in the ORNs should provide a detailed understanding of how nonvisual arrestins maintain the sensitivity of early events in olfactory signal transduction (Ge, 2006).

Regulation of Notch signalling by non-visual ß-arrestin: a trimolecular interaction between Notch, Deltex, and Kurtz

Signalling activity of the Notch receptor, which plays a fundamental role in metazoan cell fate determination, is controlled at multiple levels. A Notch signal-controlling mechanism was uncovered that depends on the ability of the non-visual ß-arrestin, Kurtz (Krz), to influence the degradation and, consequently, the function of the Notch receptor. Krz was identified as a binding partner of a known Notch-pathway modulator, Deltex (Dx), and the existence was demonstrated of a trimeric Notch-Dx-Krz protein complex. This complex mediates the degradation of the Notch receptor through a ubiquitination-dependent pathway. These results establish a novel mode of regulation of Notch signalling and define a new function for non-visual ß-arrestins (Mukherjee, 2005).

In an effort to identify elements that are integrated into the molecular circuitry affecting Notch signalling, two independent protein-interaction screens were carried out: one based on the yeast two-hybrid system, and the other based on the identification of cellular protein complexes using the tandem affinity purification (TAP)-liquid chromatography (LC)-mass spectrometry (MS)/MS approach. Both methods identified Krz as an interacting partner of Dx (Mukherjee, 2005).

A yeast two-hybrid screen was carried out using full-length Dx as bait. Eight positive clones were isolated and found to encode overlapping krz cDNAs. Sequence analysis revealed that the amino-terminal half of Krz (amino acids 10-251) is necessary and sufficient for binding Dx. The corresponding domain of mammalian non-visual ß-arrestins, which consists of the amino-terminal half of the protein, has been shown to interact with activated GPCRs (Mukherjee, 2005).

Four Krz peptides (VGEQPSIEVSK, VFELCPLLANNK, HEDTNLASSTLITNPAQR and ESLGIMVHYK) were also identified, using LC-MS/MS, among proteins in the 50,000-55,000 relative molecular mass range that co-purified with full-length amino-terminally TAP-tagged Dx (NTAP-Dx) that was isolated from stably transfected Kc167 cells. These peptides correspond to endogenous Krz protein (with a predicted relative molecular mass of 51,200) that is expressed at normal levels. Krz was also identified as a Dx-interacting partner in an independent experiment involving another cell line (S2) that was stably transfected with the NTAP-Dx transgene (Mukherjee, 2005).

To address the functional implications of the association between the Krz and Dx proteins, an investigation was carried out to see whether mutations in krz and dx display genetic interactions. Two independent loss-of-function dx alleles, dx and dxSM were used, and two independent krz loss-of-function alleles, krz1 and krz2. A transheterozygous combination of dx and krz alleles (dx/+; krz/+ females) resulted in wings that were indistinguishable from the wild type. However, reducing the dose of krz in a genetic background that further reduces or eliminates dx (in dx/Y; krz/+ males) elicited enhanced wing notching and vein thickening, compared with dx hemizygotes in a krz wild-type background. Similar results were obtained using two other dx alleles, dxENU and, importantly, a recently identified dx null allele, dx152. Given that the genetic interaction between dx and krz was observed in the absence of all dx functions, it is clear that a complete absence of dx creates a sensitized genetic background that makes development of the wing margin sensitive to a decrease in the dosage of krz (Mukherjee, 2005).

To extend the analysis of the interactions between Krz, Dx and Notch, the relative subcellular localization of these proteins was tested when they were co-expressed in cultured cells. Immunocytochemical analysis revealed that the expression of either HA-Krz or Flag-Dx alone resulted in a diffuse distribution throughout the cytoplasm. By contrast, co-expression of both proteins led to a redistribution of Krz and Dx into intracellular vesicles, where they co-localized. Colocalization of co-expressed Krz and Dx was also observed in vivo in the wing imaginal discs. The nature of these vesicles remains to be determined, but several known intracellular trafficking markers (which label early and late endosome compartments, the Golgi apparatus and the endoplasmic reticulum) did not seem to co-localize with the Krz and Dx proteins (Mukherjee, 2005).

To probe the functional significance of an interaction between Krz, Dx and Notch in vivo, the effects of krz loss of function on the endogenous Notch receptor were examined. To this end, krz loss-of-function clones were generated in two different tissues, the wing and eye-antennal imaginal discs, using the krz1-null mutant and the FLP/FRT system. It was found that the levels of the Notch protein, normally expressed throughout these discs, were substantially elevated in krz mutant cells compared with the surrounding wild-type cells. This increased level of Notch was observed in both the wing and eye-antennal discs. It is noted, however, that in the eye discs, this elevated level of Notch was more prominent in krz clones that were located anterior to the morphogenetic furrow. In contrast with the upregulation of Notch, the levels of Dx were unaltered in krz mutant clones (Mukherjee, 2005).

The present study has revealed the existence of a hitherto unknown Notch-signal controlling mechanism that relies on modulating Notch-receptor levels through the activity of the krz gene that encodes the single non-visual ß-arrestin in Drosophila. Consequently, this analysis unveils a new role of ß-arrestins as regulators of Notch signalling. Mammalian non-visual ß-arrestins were originally thought to function exclusively in the desensitization and clathrin-mediated internalization of GPCRs. The range of ß-arrestin activity has been recently extended by uncovering their involvement in the regulation of other receptor systems. The data presented in this study further extend the spectrum of ß-arrestin functions, given the demonstration that the Drosophila non-visual ß-arrestin, Krz, can modulate the protein levels of the Notch receptor and, consequently, Notch signalling. This analysis indicates that the interaction between Krz and Notch is mediated by Dx (Mukherjee, 2005).

The biochemical nature of Dx and its full spectrum of activities are not yet fully understood. dx was first implicated in Notch signalling as a modifier of Notch phenotypes. Indirect evidence implied that Dx may have a role in the transcriptional regulation of Notch targets. Additional studies postulated that dx may define a node in the Notch-signalling pathway that is independent of Suppressor of Hairless (CBF1 in mammals), the classical effector of Notch signals. In mammals, Deltex seems to be an antagonist of Notch signals. However, overexpression of Dx in Drosophila can mimic the phenotypes that are associated with Notch gain-of-function mutations, and loss of dx function results in wing-margin phenotypes that are reminiscent of loss of Notch function, indicating a positive rather than a negative role in Notch signalling. Although the current data do not exclude the possibility that Dx may have a positive role in Notch signalling in certain cellular contexts, the evidence presented in this study unambiguously demonstrates that Dx, in combination with Krz, functions as a negative regulator of Notch. The results of the present analysis, together with the previously published genetic studies, indicate that Dx may behave both as an agonist and as an antagonist of Notch signalling, depending on the specific cellular context (Mukherjee, 2005).

Notwithstanding the lack of direct evidence regarding the biochemical nature of Dx, the fact that Dx contains a RING-H2 and two WWE domains indicates that Dx may function as an E3 ubiquitin ligase. In fact, E3 ubiquitin-ligase activity has been shown to exist for mammalian homologues of Drosophila Dx. Mammalian ß-arrestins have also been implicated in receptor ubiquitination events. A stable association between ß-arrestins and Class B GPCRs was shown to promote receptor ubiquitination and degradation by recruiting E3 ubiquitin ligases, such as Mdm2, to the receptor (Mukherjee, 2005).

This study reproducibly observed a small increase of Notch ubiquitination in the presence of Dx, which was further enhanced following addition of Krz. Previous studies implicated Notch in both poly- and monoubiquitination events. No increase was detected in Notch monoubiquitination following addition of Dx, Krz or both, so an increase in ubiquitination is attributed to polyubiquitination of Notch. This study, therefore, associated the formation of the Notch-Dx-Krz complex with polyubiquitination of the Notch receptor and a subsequent reduction of Notch levels, apparently via proteasomal degradation. The underlying mechanism is unknown at this point, but it is possible that the incorporation of Krz into the Notch-Dx-Krz complex may promote polyubiquitination of Notch by facilitating the ubiquitin-ligase activity of Dx, by recruiting additional E3 ligases or perhaps by inducing an altered conformation of the Notch receptor (Mukherjee, 2005).

It is worth mentioning that additional E3 ligases, such as Suppressor of deltex [Su(dx)] and Nedd4, have been associated with Notch signalling. However, no co-localization of Su(dx) with vesicles containing Krz and Dx was observed following co-transfection of these three proteins in S2 cells. The data support a connection between the formation of the Notch-Dx-Krz complex and the proteasomal rather than the lysosomal degradative pathway. However, an involvement of the Krz-Dx vesicles in the intracellular trafficking of the Notch receptor cannot be excluded, despite the fact that marker analysis has not revealed the identity of these vesicles (Mukherjee, 2005).

It has been documented that non-visual ß-arrestins are involved in trafficking of GPCRs and other types of receptors. Given that krz seems to be the only ß-arrestin in the Drosophila genome, the question is raised as to whether other, non-seven-transmembrane-receptor systems are affected in krz mutant cells. It was asked whether, similar to the Notch receptor, the levels of Frizzled or the epidermal growth factor receptor (EGFR) are affected in loss-of-function krz clones in the wing or the eye imaginal discs, and no change was found in their levels or localization. However, these observations do not exclude the possibility that krz is still involved in the regulation of these and other receptors, as is the case in mammals. If, in mammalian systems, Notch is regulated by a similar mechanism, then loss-of-function mutations in ß-arrestins may result in the upregulation of Notch signals in certain tissues. This would be particularly significant in tissues in which Notch activation has a role in tumorigenesis (Mukherjee, 2005).

Together, the loss-of-function and the complementary gain-of-function analyses indicate that Krz in involved in the regulation of Notch signalling. It is proposed that one of the biological functions of Krz is to modulate the level of the Notch receptor in the cell and thereby to optimize the amount of Notch that can participate in signalling. Such regulation of Notch by Krz is likely to be, at least in part, constitutive and may not require its interaction with a ligand (Mukherjee, 2005).

It seems unlikely that the action of the Drosophila ß-arrestin Krz is confined to the Notch signalling pathway, but further studies will be necessary to establish the spectrum of Krz function. An association of other signalling receptors with Krz would not only link them to non-visual ß-arrestin function, but it would also provide a potential mode of cross-talk with Notch. These links may be important for defining the cellular framework within which controlling mechanisms have evolved to act on evolutionarily conserved signalling pathways such as Notch (Mukherjee, 2005).

kurtz, a novel nonvisual arrestin, is an essential neural gene in Drosophila

The kurtz gene encodes a novel nonvisual arrestin. krz is located at the most-distal end of the chromosome 3R, the third gene in from the telomere. krz is expressed throughout development. During early embryogenesis, krz is expressed ubiquitously and later is localized to the central nervous system, maxillary cirri, and antennal sensory organs. In late third instar larvae, krz message is detected in the fat bodies, the ventral portion of the thoracic-abdominal ganglia, the deuterocerebrum, the eye-antennal imaginal disc, and the wing imaginal disc. The krz1 mutation contains a P-element insertion within the only intron of this gene and results in a severe reduction of function. Mutations in krz have a broad lethal phase extending from late embryogenesis to the third larval instar. The fat bodies of krz1 larva precociously dissociate during the midthird instar. krz1 is a type 1 melanotic tumor gene; the fat body is the primary site of melanotic tumor formation during the third instar. These phenotypes have been functionally rescued with both genomic and cDNA transgenes. Importantly, the expression of a full-length krz cDNA within the CNS rescues the krz(1) lethality. These experiments establish the krz nonvisual arrestin as an essential neural gene in Drosophila (Roman, 2000). Full text of article.


Functions of Kurtz orthologs in other species

β-Arrestin-dependent dopaminergic regulation of Calcium channel activity in the axon initial segment

G-protein-coupled receptors (GPCRs) initiate a variety of signaling cascades, depending on effector coupling. β-arrestins, which were initially characterized by their ability to "arrest" GPCR signaling by uncoupling receptor and G protein, have recently emerged as important signaling effectors for GPCRs. β-arrestins (see Drosophila Kurtz) engage signaling pathways that are distinct from those mediated by G protein. As such, arrestin-dependent signaling can play a unique role in regulating cell function, but whether neuromodulatory GPCRs utilize β-arrestin-dependent signaling to regulate neuronal excitability remains unclear. In a study of cartwheel cells, a class of glycinergic interneuron in the auditory brainstem dorsal cochlear nucleus (DCN), D3 dopamine receptors (D3R) were found to regulate axon initial segment (AIS) excitability through β-arrestin-dependent signaling, modifying CaV3 voltage dependence to suppress high-frequency action potential generation. This non-canonical D3R signaling thereby gates AIS excitability via pathways distinct from classical GPCR signaling pathways (Yang, 2016) .

GPCR-G protein-β-Arrestin super-complex mediates sustained G protein signaling

Classically, G protein-coupled receptor (GPCR) stimulation promotes G protein signaling at the plasma membrane, followed by rapid β-arrestin-mediated desensitization and receptor internalization into endosomes. However, it has been demonstrated that some GPCRs activate G proteins from within internalized cellular compartments, resulting in sustained signaling. This study used a variety of biochemical, biophysical, and cell-based methods to demonstrate the existence, functionality, and architecture of internalized receptor complexes composed of a single GPCR, β-arrestin, and G protein. These super-complexes or "megaplexes" more readily form at receptors that interact strongly with β-arrestins via a C-terminal tail containing clusters of serine/threonine phosphorylation sites. Single-particle electron microscopy analysis of negative-stained purified megaplexes reveals that a single receptor simultaneously binds through its core region with G protein and through its phosphorylated C-terminal tail with β-arrestin. The formation of such megaplexes provides a potential physical basis for the newly appreciated sustained G protein signaling from internalized GPCRs (Thomsen, 2016).


REFERENCES

Search PubMed for articles about Drosophila Kurtz

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Chen, W., et al. (2004). Activity-dependent internalization of Smoothened mediated by beta-arrestin 2 and GRK2. Science 306: 2257-2260

Cheng, S., Maier, D., Neubueser, D. and Hipfner, D. R. (2010). Regulation of Smoothened by Drosophila G-protein-coupled receptor kinases. Dev. Biol. 337: 99-109. PubMed ID: 19850026

Conner, D. A., et al. (1997). beta-Arrestin1 knockout mice appear normal but demonstrate altered cardiac responses to beta-adrenergic stimulation. Circ. Res. 81(6): 1021-6. PubMed ID: 9400383

Craft, C. M. and Whitmore, D. H. (1995). The arrestin superfamily: cone arrestins are a fourth family. FEBS Lett. 362: 247-255. PubMed ID: 7720881

Fukuto, H. S., et al. (2004). G protein-coupled receptor kinase function is essential for chemo-sensation in C. elegans. Neuron 42: 581-593. PubMed ID: 15157420

Ge, H., et al. (2006). A Drosophila nonvisual arrestin is required for the maintenance of olfactory sensitivity. Chemical Senses 31: 49-62. PubMed ID: 16306316

Gurevich, V. V., et al. (1995). Arrestin interactions with G protein-coupled receptors. Direct binding studies of wild type and mutant arrestins with rhodopsin, beta 2-adrenergic, and m2 muscarinic cholinergic receptors. J. Biol. Chem. 270: 720-731. PubMed ID: 7822302

Gurevich, V. V. and Gurevich, E. V. (2004). The molecular acrobatics of arrestin activation. Trends Pharmacol Sci 25: 105-111. PubMed ID: 15102497

Johnson, E. C., Tift, F. W., McCauley, A., Liu, L. and Roman, G. (2008). Functional characterization of kurtz, a Drosophila non-visual arrestin, reveals conservation of GPCR desensitization mechanisms. Insect Biochem. Mol. Biol. 38: 1016-1022. PubMed ID: 18938246

Kovacs, J. J., et al. (2009). Arrestin development: emerging roles for beta-arrestins in developmental signaling pathways. Dev. Cell 17: 443-458. PubMed ID: 19853559

Kirchhausen, T., Bonifacino, J. S. and Riezman, H. (1997). Linking cargo to vesicle formation: receptor tail interactions with coat proteins. Curr. Opin. Cell Biol. 9: 488-495. PubMed ID: 9261055

Krueger, K. M., Daaka, Y., Pitcher, J. A. and Lefkowitz, R. F. (1997). The role of sequestration in G-protein coupled receptor resensitization: regulation of β2-adrenergic receptor dephosphorylation by vesicular acidification. J. Biol. Chem. 272: 5-8. PubMed ID: 8995214

Lefkowitz, R. J. and Whalen, E. J. (2004). beta-arrestins: traffic cops of cell signaling. Curr. Opin. Cell Biol. 16(2): 162-8. PubMed ID: 15196559

Lefkowitz R. J. and Shenoy, S. K. (2005). Transduction of receptor signals by beta-arrestins. Science 308: 512-517. PubMed ID: 15845844

Li, S., Chen, Y., Shi, Q., Yue, T., Wang, B. and Jiang, J. (2012). Hedgehog-regulated ubiquitination controls smoothened trafficking and cell surface expression in Drosophila. PLoS Biol 10: e1001239. PubMed ID: 22253574

Liu, L., Davis, R. L. and Roman, G. (2007). Exploratory activity in Drosophila requires the kurtz nonvisual arrestin. Genetics 175: 1197-1212. PubMed ID: 17151232

Meloni, A. R., et al. (2006) Smoothened signal transduction is promoted by G protein-coupled receptor kinase 2. Mol Cell Biol 26: 7550-7560. PubMed ID: 16908539

Mishra, A. K., Sachan, N., Mutsuddi, M. and Mukherjee, A. (2014). TRAF6 is a novel regulator of Notch signaling in Drosophila melanogaster. Cell Signal 26: 3016-3026. PubMed ID: 25280943

Molnar, C., Ruiz-Gómez, A., Martín, M., Rojo-Berciano, S., Mayor, F. and de Celis, J. F. (2011). Role of the Drosophila non-visual β-arrestin kurtz in hedgehog signalling. PLoS Genet. 7(3): e1001335. PubMed ID: 21437272

Mukherjee, A., et al. (2005). Regulation of Notch signalling by non-visual beta-arrestin. Nature Cell Biol 7: 1191-1201. PubMed ID: 16284625

Oakley, R. H., et al. (1999). Association of beta-arrestin with G protein-coupled receptors during clathrin-mediated endocytosis dictates the profile of receptor resensitization. J. Biol. Chem. 274: 32248-32257. PubMed ID: 10542263

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Pitcher. J. A., et al. (1995). The G-protein coupled receptor phosphatase: a protein phosphatase type 2A with a distinct subcellular distribution and substrate specificity. Proc. Natl. Acad. Sci. 92: 8343-8347. PubMed ID: 7667292

Roman, G., He, J. and Davis, R. L. (2000). kurtz, a novel nonvisual arrestin, is an essential neural gene in Drosophila. Genetics 155: 1281-1295. PubMed ID: 10880488

Shanbhag, S. R., Müller, B. and Steinbrecht, R. A. (1999). Atlas of olfactory organs of Drosophila melanogaster. 1 Types, external organization, innervation, and distribution of olfactory sensilla. Int. J. Insect. Morphol. Embryol. 28: 377-397

Sterne-Marr R., et al. (1993). Polypeptide variants of beta-arrestin and arrestin3. J. Biol. Chem. 268: 15640-15648. PubMed ID: 8340388

Thomsen, A. R., et al. (2016). GPCR-G protein-β-Arrestin super-complex mediates sustained G protein signaling. Cell 166: 907-919. PubMed ID: 27499021

Tipping, M., et al. (2010). beta-arrestin Kurtz inhibits MAPK and Toll signalling in Drosophila development. EMBO J. 29: 3222-3235. PubMed ID: 20802461

Wilbanks, A. M., et al. (2004). Beta-arrestin 2 regulates zebrafish development through the hedgehog signaling pathway. Science 306: 2264-2267. PubMed ID: 15618520

Yang, S., Ben-Shalom, R., Ahn, M., Liptak, A. T., van Rijn, R. M., Whistler, J. L. and Bender, K. J. (2016). β-Arrestin-dependent dopaminergic regulation of Calcium channel activity in the axon initial segment. Cell Rep. PubMed ID: 27452469


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date revised: 20 October 2016

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