G protein-coupled receptor kinase 2
Alpha-synuclein is phosphorylated at serine 129 (Ser129) in intracellular protein aggregates called Lewy bodies. These inclusion bodies are the characteristic pathologic lesions of Parkinson disease. This study defines the role of phosphorylation of Ser129 in alpha-synuclein toxicity and inclusion formation using a Drosophila model of Parkinson disease. Mutation of Ser129 to alanine to prevent phosphorylation completely suppresses dopaminergic neuronal loss produced by expression of human alpha-synuclein. In contrast, altering Ser129 to the negatively charged residue aspartate, to mimic phosphorylation, significantly enhances alpha-synuclein toxicity. The G protein-coupled receptor kinase 2 (Gprk2) phosphorylates Ser129 in vivo and enhances alpha-synuclein toxicity. Blocking phosphorylation at Ser129 substantially increases aggregate formation. Thus Ser129 phosphorylation status is crucial in mediating alpha-synuclein neurotoxicity and inclusion formation. Because increased number of inclusion bodies correlates with reduced toxicity, inclusion bodies may protect neurons from alpha-synuclein toxicity (Chen, 2005).
Signaling by Smoothened (Smo) plays fundamental roles during animal development and is deregulated in a variety of human cancers. Smo is a transmembrane protein with a heptahelical topology characteristic of G protein-coupled receptors. Despite such similarity, the mechanisms regulating Smo signaling are not fully understood. Gprk2, a Drosophila member of the G protein-coupled receptor kinases, plays a key role in the Smo signal transduction pathway. Lowering Gprk2 levels in the wing disc reduces the expression of Smo targets and causes a phenotype reminiscent of loss of Smo function. Gprk2 function is required for transducing the Smo signal, and when Gprk2 levels are lowered, Smo still accumulates at the cell membrane, but its activation is reduced. Interestingly, the expression of Gprk2 in the wing disc is regulated in part by Smo, generating a positive feedback loop that maintains high Smo activity close to the anterior-posterior compartment boundary (Molnar, 2007).
Smo is the key transducer of a conserved signaling pathway regulating many developmental processes in vertebrates and invertebrates. The transmembrane protein Patched (Ptc) is the receptor for the ligand Hedgehog (Hh) and represses Smo activity in the absence of ligand. The binding of Hh to Ptc relieves this repression and allows Smo to signal to a protein complex that includes the transcription factor Ci/Gli. Smo controls the activation of Ci in the presence of the Hh ligand in part by preventing Ci proteolytic processing into a transcriptional repressor. In the Drosophila wing disc, the epithelium giving rise to the wing and thorax of the fly, Smo signaling controls the expression of several genes in anterior cells close to the anterior-posterior (A/P) compartment boundary and promotes the growth and patterning of the wing (Molnar, 2007).
The cytoplasmic tail of Drosophila Smo is a target for phosphorylation by protein kinase A and casein kinase I, and it has been shown that Smo phosphorylation by these kinases is essential for its activity and membrane accumulation. However, most of these phosphorylated residues are not conserved in its vertebrate counterparts. Recently, the G protein-coupled receptor kinase 2 (Grk2) has been shown to phosphorylate mammalian Smo (Chen, 2004). G protein-coupled receptor kinases (GRKs) selectively phosphorylate the ligand-activated form of G protein-coupled receptors (Lefkowitz, 2005). This phosphorylation promotes uncoupling from G proteins and also the recruitment of β-arrestins, which target the receptor for clathrin-mediated endocytosis. In addition, GRKs and β-arrestins also participate in signal propagation by recruiting additional proteins to the receptor complex. There are two Drosophila GRKs, 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 (Schneider, 2005; Lannutti, 2001). Phosphorylation of mammalian Smo by GRK2 promotes its endocytosis in clathrin-coated pits in a process dependent on β-arrestin2 (Chen, 2004). However, whether this form of Smo internalization is part of a desensitization mechanism, as is the case for different G protein-coupled receptors (Lefkowitz, 2005), or if it participates in Hh signaling is still not known. To address the participation of GRKs during Smo signaling in Drosophila, the function was analyzed of Gprk2 during imaginal wing disc development. It was found that Gprk2 activity is required for Smo activation. Thus, the reduction of Gprk2 expression by interference RNA, or its elimination by a genetic mutation, causes the accumulation of Smo in wing disc anterior cells exposed to Hh. The accumulation of Smo is, however, correlated with reduced activity, because Smo high-level targets are not correctly activated and flies expressing Gprk2-RNAi display Hh loss-of-function phenotypes. Interestingly, the reduction in Gprk2 expression is able to antagonize the activity of Smo mutant forms that mimic its phosphorylation by protein kinase A and casein kinase 1, suggesting that additional phosphorylation by Gprk2 is a necessary step to obtain the correct activation of Smo to promote the expression of its targets requiring high levels of signaling (Molnar, 2007).
The expression of Gprk2 mRNA in the wing disc is generalized but appears increased in a stripe of cells located close to the A/P compartment boundary. To better characterize this pattern, the P-lacZ insertion Gprk206936, which is localized in the 5' untranslated region of the gene, was used. Interestingly, β-gal expression is restricted to the A/P compartment boundary of the wing disc during the third larval instar. The cells expressing β-gal were further identified by using a combination of region-specific markers such as Engrailed (En), Patched (Ptc), Blistered (Bs), and Caupolican (Caup). This analysis places the stripe of maximal expression of Gprk2 to anterior cells abutting the A/P boundary. These cells express Ptc and En in the anterior compartment and are localized in the region exposed to high-level Hh signaling. In fact, Hh signaling regulates the expression of Gprk2 in these anterior cells, because β-gal expression in Gprk206936 discs is expanded to the entire anterior compartment when hh is ectopically expressed, and it is repressed when the activity of the pathway is reduced by ectopic expression of Ptc). The regulation of Gprk2 accumulation in anterior cells by Hh suggests that Hh signaling and Gprk2 might be functionally related (Molnar, 2007).
All available Gprk2 alleles are P element insertions in the 5' region of the gene. These alleles are homozygous viable, and the mutant wings do not display any visible phenotype. Stronger loss-of-function conditions of the gene were generated by (1) expressing Gprk2 interference RNA (Gprk2i) under the control of yeast upstream activator sequences (UAS; UAS-Gprk2i) and (2) constructing a synthetic deletion of the gene. In wing discs of the combination Gal4-638/UAS-Gprk2i, a reduction of Gprk2 mRNA levels of 66% was found. The corresponding adult wings show a range of striking phenotypes similar to loss of Hh function, displaying a reduction of the L3/L4 intervein, fusion of the L3 and L4 veins, and in a lower percentage of wings, the loss of the L3 and L4 veins. These veins and the L3/L4 intervein correspond to the territory specified by Hh signaling. In fact, reduction of Gprk2 levels results in wings very similar to those with a moderate loss of Hh signaling, generated either by ectopic expression of Ptc or by expression or hh-interference RNA. This phenotype is very different from that observed upon increased activity of the pathway. The Gal4-638 line is expressed in the entire wing, and to distinguish between the effects of lowering Gprk2 levels in cells producing or responding to Hh, three other Gal4 lines were used expressed in either the anterior (Gal4-Ci and Gal4-ptc) or the posterior (Gal4-hh) compartments. It was found that the expression of Gprk2i only in anterior cells recapitulates the reduction of the L3/L4 intervein observed in Gal4-638/UAS-Gprk2i wings. Thus, the combinations Gal4-Ci/UAS-Gprk2i and Gal4-ptc/UAS-Gprk2i show a reduction or elimination of the L3/L4 intervein, whereas the wings of the Gal4-hh/UAS-Gprk2i combination display a normal pattern of veins. The phenotypes observed upon a reduction of Gprk2 unambiguously indicate that Gprk2 function is necessary for the transduction of the Hh signal. Furthermore, when the expression of Gprk2 is reduced in flies expressing lower levels of the ligand Hh, the resulting wings have stronger hh loss-of-function phenotypes, and a previously unrecognized phenotypic class indistinguishable to those of wings formed by smo mutant cells is now observed (Molnar, 2007).
To directly monitor the activity of the Hh pathway, the expression of several Hh targets was studied in Gal4-638/UAS-Gprk2i discs. The expression of En and Ptc in anterior cells is always impaired when Gprk2 levels are reduced. These two genes correspond to Hh targets activated by a high level of signaling. The expression of Knot (Kn) is also reduced in Gal4-638/UAS-Gprk2i discs, and the stripe of maximal accumulation of Ci is also modified in Gal4-638/UAS-Gprk2i discs compared with wild-type ones. The expression of other genes regulated directly or indirectly by Hh signaling was studied in Gal4-638/UAS-Gprk2i discs. The expression of the Notch ligand Delta (Dl) is very weak or absent in the primordia of the veins L3 and L4, where it accumulates at high levels in normal discs. Similarly, the expression of Bs in the L3/L4 intervein is reduced or absent in Gal4-638/UAS-Gprk2i discs. The expression of the low-level Hh signaling targets caup and decapentaplegic (dpp) is also modified in Gal4-638/UAS-Gprk2i discs. Caup expression in the presumptive L3 vein is generally expanded toward the A/P compartment boundary in Gal4-638/UAS-Gprk2i discs, most likely because En, a repressor of Caup in anterior cells, is not expressed upon a reduction of Gprk2 levels. The domain of Caup expression in the L3 vein is reduced or lost compared with wild-type discs in only a small fraction of discs (7%). The expression of dpp is detected in Gal4-638/UAS-Gprk2i discs at lower levels but in a domain broader than the characteristic of normal discs. Taken together, these data suggest that Gprk2 plays a positive role in the Hh signaling pathway. The lowering of Gprk2 levels reduces very efficiently high-level Hh signaling and much less efficiently low-level Hh signaling. Thus, a complete elimination of Hh signaling is observed only when Gprk2 levels are reduced in wing discs with lower hh. Finally, the expression of spalt, a target of the Dpp/BMP4 pathway, is almost normal upon Gprk2 reduction, indicating specificity of Gprk2 function toward Hh signaling (Molnar, 2007).
To confirm the specificity of the Gprk2 RNAi, the expression of two Hh-targets, En and Ptc, was analyzed in wing disc cells homozygous for a deficiency that removes all of the Gprk2 coding region. In both cases it was found that anterior Gprk2 - clones eliminate, in a cell-autonomous manner, the anterior expression of En. Gprk2 - clones located in the posterior compartment did not affect the expression of En, confirming that Gprk2 activity is required in cells receiving Hh (Molnar, 2007).
To further analyze where Gprk2 function is required in the Hh signaling pathway, the expression of En was studied in clones of cells ectopically expressing hh or both hh and Gprk2i. It was found that clones expressing Gprk2i located in the domain of En expression in the anterior compartment cell-autonomously suppress the expression of En. The expression of En is induced by Hh signaling in hh-expressing clones, both within the clone and in the surrounding cells. However, in the hh+Gprk2i-expressing clones, the expression of En is induced only in wild-type anterior cells that do not express Gprk2i. These observations confirm that Gprk2 activity is required for transducing the Hh signal in Hh-receiving cells and not for Hh secretion (Molnar, 2007).
Experiments in mammalian cells in culture have shown that beta-arrestin2 and GRK2 mediate internalization of active Smo (Chen, 2004). Consequently, the expression and subcellular localization of Smo was studied in wing discs where Gprk2 activity is reduced. In wild-type discs, smo RNA is expressed in all cells, but Smo protein accumulates associated to cell membranes only in the posterior compartment and in some anterior cells exposed to Hh. Intriguingly, the reduction in Gprk2 levels in the entire wing blade eliminates the distinction in Smo accumulation between anterior and posterior cells, and Smo is detected at similar levels in both compartments. When the levels of Gprk2 are reduced only in the dorsal compartment or in clones of Gprk2 - homozygous cells, the changes in Smo expression in anterior cells are more evident. Thus, it was observed that Smo accumulates at high levels associated to cell membranes in a broader anterior domain of cells within the range of Hh. The extension of Smo accumulation in anterior cells might be due to an extension of the Hh diffusion range because Ptc is not expressed in Gprk2 mutant cells. This is a previously unrecognized instance in which Smo accumulation and signaling can be uncoupled, because it was thought that, at least in Drosophila, Smo membrane accumulation leads to signaling. The same effects are observed when S2 cells were used. Thus, Smo is expressed in S2 cells in intracellular vesicles at low levels. Upon Hh treatment, Smo translocates close to the plasma membrane in these cells. In cells that have been treated for 4 days with Gprk2 dsRNA (causing a reduction of Gprk2 mRNA levels of 77%) the levels of Smo are higher independently of Hh (Molnar, 2007).
To further analyze the relationship between Smo and Gprk2 functions, Gprk2i was expressed in the same wing with different N-terminal (smoDeltaN; extracellular) and C-terminal (smoDeltaC2 and smoDeltaC4; intracellular) deletions of Smo. The expression of Smo proteins bearing either N-terminal or C-terminal deletions fails to rescue Smo mutants, but their overexpression does not interfere significantly with Smo signaling. A strong synergic genetic interaction was found when Smo C-terminal deletions were coexpressed with Gprk2i. Thus, wings expressing C-terminal deletions of Smo with reduced Gprk2 levels display a strong hh loss-of-function phenotype that is comparable to the elimination of smo. Gprk2i combined with UAS-smoDeltaN resulted in additive phenotypes. It is suggested that the reduction of Gprk2 uncovers a dominant-negative effect of SmoDeltaC proteins, reducing the efficiency of Smo signaling. The basis for this dominant negative effect could be the inclusion of a form of Smo, SmoDeltaC, unable to be phosphorylated by Gprk2, in the Smo complexes that have been postulated to mediate Smo activity. Therefore, it is proposed that Gprk2 function, acting through the C-terminal tail of Smo, is involved in an activation step promoting Smo interaction with the Costal2/Fused/Su(fu) complex to prevent Ci processing into a repressor form and to accumulate Ci in an activating form. Based on the effects of mammalian GRK2 and beta2-arrestin on Smo (Chen, 2004, Meloni, 2006), it is possible that Gprk2-mediated activation of Smo involves the recycling of Smo from the cell membrane to an intracellular signaling compartment (Molnar, 2007).
The interaction between SmoDeltaC and Gprk2 indicates a critical role of the Smo intracellular C-terminal domain for its relationship with Gprk2 function. Interestingly, the Smo intracellular C-terminal domain is where all of the consensus phosphorylation sites by casein kinase 1 and protein kinase A are located, as well as other serine and threonine residues in the vicinity of acidic residues that are similar to mammalian GRK2 phosphorylation consensus. A form of Smo was expressed in the wing disc that mimics its phosphorylation by these kinases (SmoSD123), and whether this Smo-activated form is sensitive to Gprk2 levels was analyzed. The expression of SmoSD123 in the wing disc causes overgrowth of the anterior compartment and defects in the L3 and L2 veins. In the corresponding wing discs, the accumulation of Smo and the expression of its targets En and Ptc are expanded to occupy the entire anterior compartment. When Gprk2 levels are reduced in discs expressing SmoSD123, Smo accumulation is still observed in all anterior cells. In contrast, the expression of both En and Ptc is now restricted to their normal domains adjacent to the A/P compartment boundary. The overgrowth phenotype characteristic of Gal4-638/+; UAS-SmoSD123 discs is not rescued by the reduction of Gprk2 expression, suggesting that the low-level Hh target dpp is still expressed through the anterior compartment. These data suggest that to generate the high levels of Smo activity required to activate the expression of its targets En and Ptc, the SmoSD123 protein has to be phosphorylated by Gprk2 (Molnar, 2007).
In conclusion, Drosophila Gprk2 is critically required to generate high levels of Hh signaling in the wing disc. The genetic interactions between Gprk2 and Smo proteins bearing C-terminal deletions or Smo phosphomimic variants suggest that Smo is a target of Gprk2. The modifications in Smo protein accumulation detected in wing discs and S2 cells with reduced Gprk2 expression suggests that a likely step affected by Gprk2 is the activation of Smo by a phosphorylation step that could prime Smo for internalization to a signaling compartment. GRK2 has recently been shown to play a positive role in Shh transduction in mammalian cells (Meloni, 2006). Taken together, these findings and the data indicate that Smo phosphorylation by GRK homologues constitute a conserved component of the Smo signal transduction cascade (Molnar, 2007).
Circadian changes in membrane potential and spontaneous firing frequency have been observed in microbial systems, invertebrates, and mammals. Oscillators in olfactory sensory neurons (OSNs) from Drosophila are both necessary and sufficient to sustain rhythms in electroanntenogram (EAG) responses, suggesting that odorant receptors (ORs) and/or OR-dependent processes are under clock control. This study measured single-unit responses in different antennal sensillae from wild-type, clock mutant, odorant-receptor mutant, and G protein-coupled receptor kinase 2 (Gprk2) mutant flies to examine the cellular and molecular mechanisms that drive rhythms in olfaction. Spontaneous spike amplitude, but not spontaneous or odor-induced firing frequency, is under clock control in ab1 and ab3 basiconic sensillae and T2 trichoid sensillae. Mutants lacking odorant receptors in dendrites display constant low spike amplitudes, and the reduction or increase of levels of GPRK2 in OSNs results in constant low or constant high spontaneous spike amplitudes, respectively. It is concluded that spike amplitude is controlled by circadian clocks in basiconic and trichoid sensillae and requires GPRK2 expression and the presence of functional ORs in dendrites. These results argue that rhythms in GPRK2 levels control OR localization and OR-dependent ion channel activity and/or composition to mediate rhythms in spontaneous spike amplitude (Krishnan, 2008).
One hypothesis to explain rhythms in the amplitude of spontaneous spikes and electroanntenogram rhythms (EAGs) is that ion channel activity and/or composition is under circadian control. Drosophila ORs have been found to form heteromeric odor-gated and cyclic-nucleotide-activated cation channels. It has been demonstrated that ORs accumulate in OSN dendrites in a circadian fashion, where OR abundance peaks near the middle of the night and is low during the day (Tanoue, 2008). These rhythms are dependent on the levels of GPRK2 and coincide with rhythms in the amplitude of both EAGs and spontaneous spikes (Tanoue, 2008). Taken together, these results suggest a model whereby GPRK2 controls the abundance and/or activity of OR-dependent odor-gated cation channels in OSN dendrites, which in turn alter membrane conductance to generate rhythms in the amplitude of spontaneous spikes and EAG responses. The possibility cannot be excluded that the clock modulates other molecular or cellular targets to generate rhythms in EAG and spike amplitude such as other ion channels expressed in OSNs, the composition of sensillar lymph, and/or the size and shape of OSNs (Krishnan, 2008).
Though a clear ecological explanation for rhythms in spike amplitude is not understood, one could hypothesize that the circadian clock could tune the olfactory system to a higher gain level (higher signal-to-noise ratio) by modulating spike amplitude irrespective of the stimulus preferentially in the subjective night. For instance, rhythms in OSN spike amplitude might produce increased activity in downstream neurons during the night and decreased activity during the day in response to the same stimulus. Recent evidence demonstrates that locomotor activity of paired male and female Drosophila is increased during the subjective night and is dependant on an intact olfactory system (Fujii, 2007). In addition, behavioral responses to odors in Drosophila are lower during the day than at night and are controlled by the circadian clock (Zhou, 2005). These phenomena may represent behavioral consequences of this electrophysiological rhythm that could provide an advantage in courtship, food acquisition, or predator avoidance. These data are consistent with circadian-clock-dependent rhythms in mating activity (Sakai, 2001). The phase of the peak in spontaneous spikes in trichoids could translate to heightened behavioral activity associated with mating during the subjective night. Thus, the results suggest that spike amplitude, in addition to firing frequency, can also encode meaningful information in the peripheral OSNs, which is transmitted to higher processing centers of CNS that mediate behavioral responses to odors (Krishnan, 2008).
G protein-coupled receptor kinase 2 (Gprk2/GRK2) plays a conserved role in modulating Hedgehog (Hh) pathway activity, but its mechanism of action remains unknown. This study provides evidence that Gprk2 promotes high-level Hh signaling by regulating Smoothened (Smo) conformation through both kinase-dependent and kinase-independent mechanisms. Gprk2 promotes Smo activation by phosphorylating Smo C-terminal tail (C-tail) at Ser741/Thr742, which is facilitated by PKA and CK1 phosphorylation at adjacent Ser residues. In addition, Gprk2 forms a dimer/oligomer and binds Smo C-tail in a kinase activity-independent manner to stabilize the active Smo conformation, and promotes dimerization/oligomerization of Smo C-tail. Gprk2 expression is induced by Hh signaling, and Gprk2/Smo interaction is facilitated by PKA/CK1-mediated phosphorylation of Smo C-tail. Thus, Gprk2 forms a positive feedback loop and acts downstream from PKA and CK1 to facilitate high-level Hh signaling by promoting the active state of Smo through direct phosphorylation and molecular scaffolding (Chen, 2010).
A genetic modifier screen for novel Hh signaling components identified Gprk2 as a positive regulator of Smo. Gprk2 was shown to be required for high but not low levels of Hh signaling activity. Evidence was provided that Gprk2 is a Smo kinase and that Gprk2 promotes maximal Smo activity by phosphorylating S741/T742 in Smo C-tail. Furthermore, a kinase-independent function of Gprk2 in Hh signaling was uncovered. Gprk2 forms a dimer/oligomer and binds Smo C-tail to promote the active state of Smo. Thus, this study reveals a novel mechanism for regulating a GPCR-like protein by GRK (Chen, 2010).
Previous studies suggest that Drosophila Gprk2 and mammalian GRK2 are involved in Smo phosphorylation because their knockdown in cultured cells either increased Smo mobility on SDS-PAGE or decreased metabolic labeling of Smo by γ-32p-ATP. However, these studies did not distinguish whether Gprk2/GRK2 phosphorylates Smo directly or indirectly through regulating other kinases. Neither did they reveal any biological relevance of Gprk2/GRK2-mediated phosphorylation in Hh signaling, since the relevant phosphorylation sites on Smo were not identified. In an in vitro kinase assay using purified substrates and a recombinant GRK, this study found that Smo is phosphorylated by GRK at S741/T742 and S1013/S1015. A mutagenesis study demonstrated that phosphorylation at S741/T742 is required for optimal Smo activation. Indeed, a previous study showed that Smo is phosphorylated at S741/T742 in cultured cells in the presence of Hh. In further agreement with the functional significance of S741/T742 phosphorylation, conserved S/T residues are found at the corresponding location in other insect Smo proteins (FlyBase) (Chen, 2010).
Interestingly, the in vitro kinase assay revealed that phosphorylation of S741/T742 by Gprk2 is regulated by PKA/CK1 phosphorylation at adjacent Ser residues, including S740, S743, and S746. Previous studies in mammalian systems suggest that GRKs tend to phosphorylate S/T residues embedded in an acidic environment. Phosphorylation at S740, S743, and S746 improves the acidic environment for S741/T742, which may account for the observed stimulation of S741/T742 phosphorylation by PKA/CK1. Indeed, mutating S740, S743, and S746 to Ala abolished PKA/CK1-mediated stimulation of S741/T742 phosphorylation, whereas converting these residues to acidic residues mimicked PKA/CK1-mediated stimulation. As Hh induces Smo phosphorylation by PKA and CK1, phosphorylation at S741/T742 by Gprk2 is likely to be stimulated by Hh in vivo (Chen, 2010).
Although phosphomimetic mutation at S741/T742 promotes Smo activity, it does not bypass the requirement for Gprk2 for optimal Smo activation because SmoSDGPSD failed to induce ectopic en expression in Gprk2 mutant discs. This implies that Gprk2 promotes Hh signaling through a mechanism in parallel to S741/T742 phosphorylation. It is possible that Gprk2 might act at an additional step downstream from Smo activation by phosphorylating intracellular Hh signaling components, or at the level of Smo activation by phosphorylating Smo at additional sites that have been missed by the in vitro kinase assay. However, the finding that the constitutively active form of Smo lacking the autoinhibitory domain (SAID: SmoΔ661-818) is insensitive to Gprk2 inactivation suggests that Gprk2 acts mainly at the level of Smo, although the possibility cannot be ruled out that Gprk2 may also play a minor role downstream from Smo. Interestingly, it was found that the kinase-dead form of Gprk2 (Gprk2KM) can rescue the activity defect of SmoSDGPSD in Gprk2 mutants, demonstrating that Gprk2 also regulates Smo in a phosphorylation-independent manner. The observation that Gprk2KM does not rescue the activity defect of SmoSD123 in Gprk2 mutants suggests that the phosphorylation-dependent and phosphorylation-independent mechanisms act in parallel rather than redundantly to promote Smo activation. Furthermore, evidence was obtained that Gprk2 interacts with the SAID independently of its kinase activity. Therefore, it is proposed that Gprk2 promotes Smo activation by counteracting Smo autoinhibition through binding to and phosphorylating the SAID (Chen, 2010).
At least two paralleled mechanisms have been attributed to Smo activation by Hh: (1) Smo cell surface accumulation, and (2) conformation change in Smo C-tail. Intriguingly, it was found that loss of Gprk2 resulted in increased rather than decreased Smo levels in cells that are not exposed to Hh or are exposed to low levels of Hh. However, unlike Hh stimulation, which preferentially stabilizes Smo on the cell surface, Gprk2 inactivation appears to stabilize Smo both inside the cell and on the cell surface. Furthermore, in the presence of high levels of Hh where Smo is accumulated at high levels on the cell surface, Gprk2 inactivation does not cause any discernible changes in either the level or subcellular distribution of Smo. Thus, the reduced Smo activity in Gprk2 mutant cells exposed to high levels of Hh is unlikely to be due to a change in Smo level or subcellular localization (Chen, 2010).
It is not clear what role Gprk2-mediated down-regulation of Smo levels might play in Hh signaling, although this may reflect an ancient mechanism by which GRK family kinases 'desensitize' GPCRs. In this regard, Gprk2-mediated down-regulation could serve as a mechanism to restrict the basal level of Hh signaling activity or to terminate or tune down Hh signaling activity once the Hh signal is withdrawn. However, this negative role of Gprk2 could be masked by its positive role. The mechanism by which Gprk2 down-regulates Smo levels remains unclear, although the kinase activity of Gprk2 appears to be required. Gprk2 could phosphorylate Smo and/or other proteins to promote Smo internalization and degradation. High levels of Hh could counteract Gprk2-mediated down-regulation of Smo by preventing Gprk2-meidated Smo internalization or by promoting Smo recycling (Chen, 2010).
FRET analysis provided strong evidence that Gprk2 is required for Smo to adopt and/or maintain its active conformation in response to Hh stimulation. A previous study suggested that Hh induces a conformational switch in Smo C-tail that is mediated by PKA and CK1 phosphorylation. In the absence of Hh, the Smo C-tail adopts a closed conformation in which the tail folds back, resulting in a close proximity between the C terminus and the third intracellular loop. The closed conformation is maintained, at least in part, through intramolecular electrostatic interactions between the multiple Arg clusters in the SAID and multiple acidic clusters near the C terminus. Hh-induced phosphorylation at PKA and CK1 sties disrupts the intramolecular electrostatic interactions, resulting in unfolding of the C-tail, which is reflected by a decreased intramolecular FRET (FRETL3C). In addition, phosphorylation promotes dimerization of two C-tails within a Smo homodimer, leading to increased proximity of the two C termini, as reflected by an increased C-terminal FRET (FRETC). Multiple intermediate conformational states may exist, depending on the levels of Smo phosphorylation, as increasing the number of phosphomimetic mutations progressively decreased FRETL3C and gradually increased FRETC. It was found that both an Hh-induced decrease in FRETL3C and an Hh-induced increase in FRETC were compromised by loss of Gprk2, suggesting that Gprk2 is critical for Smo to adopt and/or maintain the fully open conformation (Chen, 2010).
How does Gprk2 regulate Smo conformation? Genetic and FRET analyses demonstrated that Gprk2 promotes high levels of Hh signaling activity and regulates Smo conformation through both phosphorylation-dependent and phosphorylation-independent mechanisms. Furthermore, this study found that Gprk2 self-associates, binds the SAID, and promotes self-association of Smo C-tail. Interestingly, both Gprk2/SAID interaction and S741/T742 phosphorylation by Gprk2 are enhanced by PKA/CK1 phosphorylation. Taken together, the following model is proposed to account for the regulation of Smo conformation by Gprk2. In response to Hh, PKA/CK1-mediated phosphorylation of Smo C-tail promotes its unfolding and dimerization; however, in the absence of Gprk2, the open conformational state of Smo is unstable and may exist in equilibrium with the closed and/or partially open conformational states. Phosphorylation of Smo by PKA/CK1 promotes the binding of Gprk2 to the SAID and phosphorylation at S741/T742, both of which may stabilize Smo in the fully open and active conformation by preventing refolding of Smo C-tail and by 'cross-linking' the two C-tails within a Smo dimer via dimerization of Gprk2. In essence, Gprk2 may function as a 'molecular clamp' to promote the clustering of Smo C-tails. It is also possible that Gprk2 could cross-link two or more Smo dimers to form high-order oligomers, which might be essential for high levels of Hh signaling activity. This study thus reveals an unanticipated complexity in the regulation of Smo conformational states, and provides the first evidence that Smo conformation states are regulated by not only phosphorylation and intramolecular interactions, but also intermolecular interactions. It is possible that the closed conformation state of Smo is also regulated by intermolecular interactions in addition to intramolecular interactions. For example, it has been shown that Fu can directly bind the Smo C terminus in the absence of Hh, and this interaction may help stabilize the closed conformation of Smo C-tail. Indeed, disrupting Smo/Fu interaction led to increased basal activity of Smo (Chen, 2010).
Recent studies have emphasized the differences between vertebrate and Drosophila Hh signaling mechanisms. The sequence divergence between Drosophila and vertebrate Smo proteins and the lack of conserved PKA/CK1 phosphorylation sites in vertebrate Smo proteins have led to the proposal that vertebrate Smo proteins are activated through fundamentally distinct mechanisms. Nevertheless, a previous study revealed that Shh induces a conformational change in mSmo similar to that of dSmo, and forced clustering of mSmo also leads to pathway activation). GRK2 has been implicated as a positive regulator of the Shh pathway, and mSmo phosphorylation is affected by GRK2 silencing, although direct phosphorylation of mSmo by GRK2 has not been demonstrated. It is possible that GRK2 may substitute the role of PKA and CK1 and act as a major Smo kinase in vertebrates to promote the active Smo conformation. Alternatively, GRK2 may act in conjunction with other GRKs and/or yet-to-be-identified kinases to regulate Smo conformation, subcellular localization, and activity in vertebrates. The relatively weak phenotypes exhibited by GRK2 mutants are consistent with the latter possibility. This study also raised an interesting possibility that GRK2 may regulate mSmo not only by phosphorylation, but also by a kinase-independent mechanism such as a protein-protein interaction. Further investigation of the mechanism by which GRK2 and other kinases regulate mSmo will shed an important light on how vertebrate Smo activation is achieved (Chen, 2010).
A developmental analysis of expression has shown that the 4.0 and 5.0 kb transcripts are abundantly expressed in 0-3 hour embryos, as a result of maternal expression. The 5.0 kb transcript is no longer detectable after 0-3 hours while the 4.0 kb transcript continues to be expressed at a low level. The 5.5 kb transcript is expressed at a low level in early embryogenesis, with an increase in expression from 12 to 24 hours. Thus the three transcripts are differentially expressed during development (Schneider, 1997).
The expression of Gprk2 protein was analyzed using a polyclonal antibody. This antibody was generated against a bacterially expressed protein that contained the final 145 amino acids of the kinase domain and the entire 139 amino acid carboxyl region (Cassill, 1991). DNA probes from the corresponding genomic region hybridize to all three transcripts. In immunoblot analysis of adult tissues, a single band of 80 kDa was detected in ovaries and whole females, in close agreement to the 80.7 kDa protein predicted by the B6936 cDNA. This band is absent in ovaries from gprk26936 homozygotes whereas expression in whole females remains. Thus, as expected, Gprk2 is expressed in the ovary and its expression is disrupted in the gprk26936 mutant. In larval tissues, an 80 kDa band was observed in both the central nervous system (CNS) and in carcasses (the entire animal minus the CNS). In homozygous gprk26936 larvae, expression is no longer detectable in CNS tissue but appears to be unaffected in carcasses. These results demonstrate that gprk26936 selectively disrupts Gprk2 expression in some but not all tissues (Schneider, 1997).
The tissue distribution of the protein was studied by staining whole-mount tissues with the same antibody. The anti-Gprk2 antibody labels developing wild-type egg chambers, and most of the staining is eliminated in gprk26936 mutant ovaries. Expression is first seen in region 2B of the germarium, the stage where germline cysts are being enveloped by follicle cells. In early egg chambers, membranes between adjacent nurse cells and between nurse cells and the oocyte are labeled. In addition, staining around the germinal vesicle is sometimes observed. Membrane-associated staining persists through stage 6 but decreases in intensity at stage 7, except between the nurse cells and the oocyte. In these early stages no staining is observed in follicle cells or in the region of the nurse cells that lies adjacent to the follicle cells. During stages 8-11 membrane-associated staining decreases in the nurse cells and appears around the entire circumference of the oocyte. Weak cytoplasmic staining is observed in nurse cells and follicle cells of these stages; however, the cytoplasmic (and germinal vesicle) staining sometimes persists in gprk26936 ovaries. Thus Gprk2 protein appears to be preferentially associated with nurse cell and oocyte plasma membranes during much of oogenesis. Although in situ hybridization studies also detected GPK2 RNA only in the nurse cells, a lower level of expression in somatic cells can not be ruled out by these experiments (Schneider, 1997).
Specific staining using anti-Gprk2 is also detected in non-ovarian tissues, consistent with a role for this gene outside of the ovaries. The central nervous system (CNS) of both larvae and adults stains intensely. In larvae, staining is present in axon fascicles, especially large ones, including nerves projecting to the optic lobes, the longitudinal connectives, and portions of the mushroom bodies. Little staining is detected in cell bodies within the CNS. However, strong staining is consistently observed in the cell bodies and nerves of the corpus allatum of the ring gland. In the adult CNS, staining is restricted to two major structures within the brain. (1) The nerves within and projecting to the ellipsoid body of the central complex are consistently stained. The ellipsoid body contributes to higher brain function in flies, such as locomotion. (2) Strong staining is also observed in the mushroom bodies that included the Kenyon cells and nerve processes within the peduncles and the alpha lobes. The mushroom bodies have been implicated in memory and learning. Interestingly, the learning mutants dunce, rutabaga, and DCO, which all disrupt genes involved in G protein signaling, are predominately expressed in the mushroom bodies. In the mutant, Gprk2 staining is abolished from both the larval and adult CNS, and from the larval ring gland (Schneider, 1997).
Most of the other tissues in the larva and adult are only stained weakly and it is not possible to determine if staining is affected in the mutant. One exception to this is in the wing imaginal disc where staining is observed in the notum and in a stripe that parallels the anterior/posterior boundary of the wing blade. Within this stripe, staining is always weakest in the region corresponding to the tip of the wing. This staining is eliminated in Gprk26936 wing discs (Schneider, 1997).
The disc staining was particularly interesting because dpp is also expressed along the anterior/posterior boundary of the wing disc. To determine how Gprk2 staining corresponds to the anterior/posterior boundary, expression of Gprk2 was compared with Engrailed (En) and dpp. En protein, which is expressed in the posterior compartment of the wing disc, was visualized with a monoclonal antibody. dpp expression was visualized by ß-gal immunoreactivity in flies bearing a dpp-lacZ fusion gene. Gprk2 and dpp are co-expressed along the anterior/posterior boundary with two exceptions. (1) The stripe of Gprk2 staining is thinner, about one cell in thickness as opposed to a 2-3 cell thickness of dpp expression. The stripe of Gprk2 expression coincides with the dpp-expressing cells closest to the boundary. (2) Near the wing tip, dpp is not expressed in the cells immediately adjacent to the boundary. In this region Gprk2 staining is present in cells that do not express dpp. At the tip of the wing En staining crosses the anterior/posterior boundary. Therefore, in this region Gprk2 and En are co-expressed whereas outside of this region the patterns of the two proteins abut one another. These results confirm that Gprk2 is expressed along the anterior side of the anterior/posterior border in a pattern that overlaps dpp (Schneider, 1997).
G protein-coupled receptor activity is controlled by a number of factors including phosphorylation by the family of G protein-coupled receptor kinases. This phosphorylation is an important first step in desensitization of the receptor. The role of G protein-coupled receptor kinases in cellular physiology has been extensively studied, but less is known about their role in development. A Drosophila G protein-coupled receptor kinase mutant (gprk26936) has developmental defects throughout the life cycle of the fly. This allows the opportunity to address G protein-coupled receptor kinase's function in vivo. Using a series of transgenic flies in which the wild type Gprk2 gene is expressed under the control of the hsp70 or germline-specific promoter, in combination with germline mosaic analysis, detailed analysis was made of the developmental stages in which Gprk2 expression is required and the tissues that must express Gprk2 for rescue of the gprk26936 mutant. These studies have shown that Gprk2 expression is required in the germline for proper formation of the anterior egg structures and for early embryogenesis. In the absence of maternal Gprk2 activity, zygotic expression affords partial rescue of egg hatching, suggesting that Gprk2 also plays an important role in late embryogenesis (Fan, 2003).
Adult viable mutations of decapentaplegic, or its putative receptor saxophone, cause homozygous females to produce short rounded eggs with abnormal anterior structures. A new female sterile mutation that effects G protein-coupled receptor kinase 2 function, fs(3)06936, has been isolated in a P element mutagenesis screen that causes similar effects on egg shape. Mature oocytes produced by homozygous fs(3)06936 females are slightly shorter and more rounded than wild type. Although positioned normally, dorsal appendages in fs(3)06936 eggs are generally shorter and broader than wild type, and the two appendages on a single egg chamber frequently differ in length. The operculum is oriented more vertically than in wild type, giving the eggs a 'square-ended' appearance, but the chorion within the operculum retains its distinctive appearance and the micropyle forms. Nurse cells often fail to completely transfer their contents into the oocyte, leaving residual material that may interfere with anterior end formation. Thus fs(3)06936 appears to affect specific aspects of egg formation without grossly altering the major pattern axes of the egg (Schneider, 1997).
Two additional ovarian defects have suggested that fs(3)06936 also functions at earlier stages of oogenesis. (1) Homozygous fs(3)06936 egg chambers degenerate during vitellogenic stages (stages 8-10A) much more frequently than expected. 26.8% of ovarioles from 4-day-old fs(3)06936 females contained a degenerating vitellogenic chamber compared to only 0.7% of wild-type ovarioles. (2) Egg chamber formation slows or ceases entirely within a significant number of fs(3)06936 ovarioles. 5.2% of the mutant ovarioles contained only 0-2 egg chambers instead of the 6-7 that are present in wild type. Germaria in such ovarioles are often smaller and thinner than in wild type, like the germaria of agametic ovarioles. Although cyst production normally declines in old females, 4-day-old wild-type females contained no similar ovarioles (Schneider, 1997).
The fs(3)06936 mutant exhibits additional defects that indicate roles for this gene outside of the ovaries. Homozygous fs(3)06936 females lay a small number of eggs, but those that are laid display a maternal effect that is partially rescued by zygotic fs(3)06936+ expression. 23.7% of embryos produced by homozygous females hatch when crossed to wild-type males, compared to only 10.3% following crosses to homozygous males. The unhatched eggs displayed a wide variety of defects including twisted gastrulation, fused adjacent segments, and perforated dorsal and ventral cuticle. These defects are more severe when the embryos lack both maternal and zygotic fs(3)06936+ function (Schneider, 1997).
The Drosophila circadian clock controls rhythms in the amplitude of odor-induced electrophysiological responses that peak during the middle of night. These rhythms are dependent on clocks in olfactory sensory neurons (OSNs), suggesting that odorant receptors (ORs) or OR-dependent processes are under clock control. Because responses to odors are initiated by heteromeric OR complexes that form odor-gated and cyclic-nucleotide-activated cation channels, whether regulators of ORs were under circadian-clock control was tested. The levels of G protein-coupled receptor kinase 2 (Gprk2) messenger RNA and protein cycle in a circadian-clock-dependent manner with a peak around the middle of the night in antennae. Gprk2 overexpression in OSNs from wild-type or cyc01 flies elicits constant high-amplitude electroantennogram (EAG) responses to ethyl acetate, whereas Gprk2 mutants produce constant low-amplitude EAG responses. ORs accumulate to high levels in the dendrites of OSNs around the middle of the night, and this dendritic localization of ORs is enhanced by GPRK2 overexpression at times when ORs are primarily localized in the cell body. These results support a model in which circadian-clock-dependent rhythms in GPRK2 abundance control the rhythmic accumulation of ORs in OSN dendrites, which in turn control rhythms in olfactory responses. The enhancement of OR function by GPRK2 contrasts with the traditional role of GPRKs in desensitizing activated receptors and suggests that GPRK2 functions through a fundamentally different mechanism to modulate OR activity (Tanoue, 2008).
Many sensory systems are regulated by the circadian clock. Various insects including flies, moths, and cockroaches show circadian rhythms in odor-dependent electrophysiological and behavioral responses. In mammals, the firing rate of isolated mouse olfactory bulb neurons is regulated by the circadian clock, as are odor-evoked brain activity waves (e.g., event-related potentials [ERPs]) in humans. Daily rhythms in neuronal activity or sensitivity have been reported for other sensory systems, such as the visual and auditory systems (Tanoue, 2008).
The circadian clock modulates olfactory responses in Drosophila: Robust electroantennogram (EAG) responses are seen during the middle of the night, and weak EAG responses are seen during the middle of the day (Krishnan, 1999). These rhythms in EAG responses are controlled by the olfactory sensory neurons (OSNs) in Drosophila, which act as independent peripheral circadian oscillators (Tanoue, 2004). Colocalization of the circadian oscillator and a rhythmic output to the OSNs indicates that the abundance and/or activity of odorant receptors (ORs) and/or OR-dependent processes are under clock control. Drosophila ORs are seven-transmembrane-domain proteins that share some structural similarities with G protein-coupled receptors (GPCRs). However, recent studies demonstrate that Drosophila ORs have an inverted membrane topology compared to canonical GPCRs and function as odor-gated and cyclic-nucleotide-activated cation channels. To understand how the clock modulates odor-dependent responses, it was determined whether factors that modulate ORs were regulated by the circadian clock (Tanoue, 2008).
G protein-coupled receptor kinases (GPRKs) and arrestins act to terminate GPCR signaling in mammals, thereby protecting cells from receptor overstimulation. GPRK-phosophorylated GPCRs are internalized by arrestin and subsequently degraded or recycled. Two Gprk genes, Gprk1 and Gprk2, have been reported in Drosophila. Gprk1 messenger RNA (mRNA) is enriched in photoreceptor cells, and expression of a Gprk1 dominant-negative mutant in photoreceptors increases the amplitude of electroretinogram (ERG) responses (Lee, 2004). Gprk2 is required for egg and wing morphogenesis, as well as embryogenesis in Drosophila (Molnar, 2007; Schneider, 1997). In mammals, seven Gprk genes are divided into three subfamilies on the basis of sequence homology: the rhodopsin kinase or visual Gprk subfamily (Gprk1 and Gprk7), the β-adrenergic receptor kinase subfamily (Gprk2 and Gprk3), and the Gprk4 subfamily (Gprk4, Gprk5, and Gprk6). Gprk3 knockout mice are unable to mediate odor-induced desensitization of odorant receptors (Peppel, 1997). In contrast, loss of Gprk2 function in C. elegans olfactory sensory neurons results in reduced chemosensory behavior, suggesting that Gprk2 is necessary for GPCR signaling (Fukuto, 2004). These results suggest that GPRKs play different roles in vertebrate and invertebrate olfaction (Tanoue, 2008).
This study reports that Gprk2 expression is regulated by circadian clocks in antennae and that GPRK2 drives circadian rhythms in olfactory responses by enhancing OR accumulation in the dendrites of basiconic sensilla. Gprk2 mRNA and protein expression levels were high around the middle of the night, coincident with the peak of olfactory responses. Flies that overexpress Gprk2 in OSNs show constant high EAG responses to ethyl acetate during 12 hr light:12 hr dark (LD) cycles and accumulate high levels of ORs in OSN dendrites, whereas hypomorphic Gprk2 mutants show constant low EAG responses to ethyl acetate during LD. On the basis of these results, it is proposed that GPRK2 mediates cycles of OR accumulation in OSN dendrites to generate rhythms in EAG responses (Tanoue, 2008).
Gprk2 is a circadian output gene whose mRNA and protein peak during the middle of the night in antennae. This phase of mRNA expression is similar to that of per, tim, and other genes driven directly by CLK-CYC binding to E-box regulatory sequences. However, CLK-CYC-dependent genes are expressed at constitutively high levels in per01 and tim01 mutants and constitutively low levels in ClkJrk and cyc01 mutants, whereas Gprk2 is expressed at low levels in per01, tim01, and cyc01 mutants. Several rhythmically expressed transcripts identified by microarray analysis of heads have low levels of expression in per01 and ClkJrk mutants, but the mechanism governing their rhythmic expression has not been explored (Tanoue, 2008).
Analysis of arrhythmic clock mutants indicates that cycling levels of Gprk2 mRNA give rise to rhythms in GPRK2 protein. The levels of GPRK2 protein cycle in phase with Gprk2 mRNA and correspond to rhythms in EAG responses. Other kinases such as Double-time (Dbt), Shaggy (Sgg)/GSK3, and Casein kinase 2 (CK2) in Drosophila are constitutively expressed proteins, whereas GPRK2 is the first example of a rhythmically expressed kinase. However, other kinases such as Erk-MAP kinase and Calcium/calmodulin-dependent protein kinase II in the chicken retina are rhythmically activated due to phosphorylation and control cGMP-gated ion channels in cone photoreceptors (Tanoue, 2008).
Cycling levels of GPRK2 are coincident with rhythms in EAG responses; GPRK2 levels and EAG responses peak around the middle of the night and are at their lowest levels during the middle of the day. When GPRK2 levels are constitutively low, as in per01, tim01, and cyc01 mutants, EAG responses are also low. In addition, levels of GPRK2 are at or below the normal wild-type trough in Gprk26936 and Gprk2EY09213 mutants and generate EAG responses that are at or below those at the wild-type trough. GPRK2 levels are barely detectable in Gprk2pj1 antennae. These results suggest that Gprk2pj1 is not a null allele and raise the possibility that a Gprk2 null mutant will lack EAG responses altogether. Such a result would demonstrate that Gprk2 is required for olfactory responses per se. In contrast, constitutive overexpression of Gprk2 produces constant high EAG responses in both wild-type and cyc01 flies, demonstrating that high levels of GPRK2 can effect high amplitude EAG responses independent of other clock-dependent factors. Taken together, these results argue that Gprk2 levels control the amplitude of EAG responses. If so, this would imply that low levels of GPRK2 present in the Gprk26936 mutant do not cycle in abundance (Tanoue, 2008).
Given that GPRK2 levels regulate the amplitude of EAG responses, what is the mechanism through which GPRK2 controls EAG response amplitude? The traditional targets of GPRKs are GPCRs. In the mammalian olfactory system, GPRK3 desensitizes ORs by triggering their internalization. These results suggest that Drosophila Gprk2 is necessary for EAG responses, and, taken together with C. elegans Gprk2 function, they indicate that GPRKs play a different role in invertebrate olfaction than in vertebrate olfaction. The subcellular localization of ORs is high in dendrites of basiconic sensilla at ZT17 and low at ZT5, but the abundance of ORs in these dendrites at ZT5 can be driven to high levels by increasing GPRK2 expression. These results support a model in which the circadian clock generates a rhythm of Gprk2 expression, which in turn generates rhythms in the amplitude of EAG responses by promoting OR accumulation (and consequently odor-gated cation-channel formation) in OSN dendrites from basiconic sensillae. GPRK2-dependent rhythms in the amplitude of spontaneous spikes are also seen in OSNs, thus demonstrating that the clock controls basic (i.e., odor-independent) properties of the OSN membrane. It is possible that the rhythmic localization of odor-gated cation channels to OSN dendrites accounts for rhythms in the amplitude of spontaneous spikes. The results can't exclude the possibility that cyclic expression of other genes also contribute to rhythms in EAG responses. mRNA cycling was tested for several genes that could potentially modulate EAG responses, including arrestin 2, Gprk1, and kurtz arrestin; arrestin 2 mRNA levels cycle, but neither Gprk1 or kurtz arrestin mRNA levels cycle. Given that microarray analysis was done on fly heads depleted of antennae, microarray analysis of antennae may reveal other rhythmically expressed genes that contribute to EAG rhythms (Tanoue, 2008).
Myc-tagged ORs did not accumulate to high levels in the dendrites of trichoid sensilla at ZT17. Trichoid sensilla have different functions than basiconic sensilla; T1 trichoid sensilla detect the pheromone 11-cis-vaccenyl acetate (cVA), whereas the basiconic sensilla recognize food and plant odors. It could be that the circadian clock regulates OSN activity differently in basiconic sensilla and trichoid sensilla, although the possibility cannot be excluded that detection of Myc-tagged ORs in dendrites failed because of low expression levels in trichoid sensillae, poor permeability of Myc antibody into trichoid sensilla, or the long, thin geometry of trichoid sensillae (Tanoue, 2008).
In summary, Drosophila Gprk2 mRNA and protein expression is under clock control in antennae. The levels of GPRK2 protein determine the amplitude of EAG responses to ethyl acetate in basiconic sensillae; high levels generate high-amplitude EAGs, and low levels produce low-amplitude EAGs. This result suggests that GPRK2 directly or indirectly enhances OR activity, in contrast to the inhibition of olfactory signaling by Gprk3 in mammals. Given that the most severe Drosophila Gprk2 mutant still produces low-amplitude EAG responses, a complete loss of Gprk2 function may lack EAG responses altogether and be required for olfaction. High levels of GPRK2 enhance OR localization to dendrites of basiconic sensillae and support a model in which rhythms in GPRK2 levels drive rhythms in OR localization to dendrites that ultimately mediates rhythms in EAG responses (Tanoue, 2008).
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date revised: 25 June 2011
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