Odorant-specific requirements for arrestin function in Drosophila olfaction

The ability to modulate olfactory sensitivity is necessary to detect chemical gradients and discriminate among a multitude of odor stimuli. Desensitization of odorant receptors has been postulated to occur when arrestins prevent the activation of downstream second messengers. A paucity of in vivo data on olfactory desensitization prompts use of Drosophila melanogaster genetics to investigate arrestins' role in regulating olfactory signaling pathways. Physiological analysis of peripheral olfactory sensitivity reveals decreased responsiveness to a host of chemically distinct odorants in flies deficient for arrestin1 (arr1), arrestin2 (arr2), or both. These phenotypes are manifest in odorant- and dose- dependent fashions. Additionally, mutants display altered adaptive properties under a prolonged exposure paradigm. Behaviorally, arr1 mutants are impaired in olfactory-based orientation towards attractive odor sources. As the olfactory deficits vary according to chemical identity and concentration, they indicate that a spectrum of arrestin activity is essential for odor processing depending upon the particular olfactory pathway involved. Arrestin mutant phenotypes are hypothesized to be a consequence of down-regulation of olfactory signaling to avoid cellular excitotoxicity. Importantly, phenotypic rescue of olfactory defects in arr1(1) mutants is achieved through transgenic expression of wild-type arr1. Taken together, these data clearly indicate that arrestins are required in a stimulus-specific manner for wild type olfactory function and add another level of complexity to peripheral odor coding mechanisms that ultimately impact olfactory behavior.     

Regulation of lipopolysaccharide‐induced inflammatory response and endotoxemia by β‐arrestins

β‐Arrestins are scaffolding proteins implicated as negative regulators of TLR4 signaling in macrophages and fibroblasts. Unexpectedly, we found that β‐arrestin‐1 (β‐arr‐1) and ‐2 knockout (KO) mice are protected from TLR4‐mediated endotoxic shock and lethality. To identify the potential mechanisms involved, we examined the plasma levels of inflammatory cytokines/chemokines in the wild‐type (WT) and β‐arr‐1 and ‐2 KO mice after lipopolysaccharide (LPS, a TLR4 ligand) injection. Consistent with lethality, LPS‐induced inflammatory cytokine levels in the plasma were markedly decreased in both β‐arr‐1 and ‐2 KO, compared to WT mice. To further explore the cellular mechanisms, we obtained splenocytes (separated into CD11^b+ and CD11^b? populations) from WT, β‐arr‐1, and ‐2 KO mice and examined the effect of LPS on cytokine production. Similar to the in vivo observations, LPS‐induced inflammatory cytokines were significantly blocked in both splenocyte populations from the β‐arr‐2 KO compared to the WT mice. This effect in the β‐arr‐1 KO mice, however, was restricted to the CD11^b? splenocytes. Our studies further indicate that regulation of cytokine production by β‐arrestins is likely independent of MAPK and IκBα‐NFκB pathways. Our results, however, suggest that LPS‐induced chromatin modification is dependent on β‐arrestin levels and may be the underlying mechanistic basis for regulation of cytokine levels by β‐arrestins in vivo. Taken together, these results indicate that β‐arr‐1 and ‐2 mediate LPS‐induced cytokine secretion in a cell‐type specific manner and that both β‐arrestins have overlapping but non‐redundant roles in regulating inflammatory cytokine production and endotoxic shock in mice. J. Cell. Physiol. 225: 406–416, 2010. © 2010 Wiley‐Liss, Inc.     

A functional role for Anopheles gambiae Arrestin1 in olfactory signal transduction

Insect sensory arrestins act to desensitize visual and olfactory signal transduction pathways, as evidenced by the phenotypic effects of mutations in the genes encoding both Arr1 and Arr2 in Drosophila melanogaster. To assess whether such arrestins play similar roles in other, more medically relevant dipterans, we examined the ability of Anopheles gambiae sensory arrestin homologs AgArr1 and AgArr2 to rescue phenotypes associated with an olfactory deficit observed in D. melanogaster arrestin mutants. Of these, only AgArr1 facilitated significant phenotypic rescue of the corresponding Drosophila arr mutant olfactory phenotype, consistent with the view that functional orthology is shared by these Arr1 homologs. These results represent the first step in the functional characterization of AgArr1, which is highly expressed in olfactory appendages of An. gambiae in which it is likely to play an essential role in olfactory signal transduction. In addition to providing insight into the common elements of the peripheral olfactory system of dipterans, this work validates the importance of AgArr1 as a potential target for novel anti-malaria strategies that focus on olfactory-based behaviors in An. gambiae.     

Insights into β2‐adrenergic receptor binding from structures of the N‐terminal lobe of ARRDC3

ARRDC3 is one of six known human α‐arrestins, and has been implicated in the downregulation of the β2‐adrenergic receptor (β2AR). ARRDC3 consists of a two‐lobed arrestin fold and a C‐terminal tail containing two PPYX motifs. In the current model for receptor downregulation by ARRDC3, the arrestin fold portion is thought to bind the receptor, while the PPXY motifs recruit ubiquitin ligases of the NEDD4 family. Here we report the crystal structures of the N‐terminal lobe of human ARRDC3 in two conformations, at 1.73 and 2.8 Å resolution, respectively. The structures reveal a large electropositive region that is capable of binding phosphate ions of crystallization. Residues within the basic patch were shown to be important for binding to β2AR, similar to the situation with β‐arrestins. This highlights potential parallels in receptor recognition between α‐ and β‐arrestins.     

Phosphoinositide and Erk signaling pathways mediate activity‐driven rodent olfactory sensory neuronal survival and stress mitigation

Olfactory sensory neurons (OSNs) are the initial site for olfactory signal transduction. Therefore, their survival is essential to olfactory function. In the current study, we demonstrated that while odorant stimulation promoted rodent OSN survival, it induced generation of reactive oxygen species in a dose‐ and time‐dependent manner as well as loss of membrane potential and fragmentation of mitochondria. The MEK‐Erk pathway played a critical role in mediating these events, as its inhibition decreased odorant stimulation‐dependent OSN survival and exacerbated intracellular stress measured by reactive oxygen species generation and heat‐shock protein 70 expression. The phosphoinositide pathway, rather than the cyclic AMP pathway, mediated the odorant‐induced activation of the MEK‐Erk pathway. These findings provide important insights into the mechanisms of activity‐driven OSN survival, the role of the phosphoinositide pathway in odorant signaling, and demonstrate that odorant detection and odorant stimulation‐mediated survival proceed via independent signaling pathways. This mechanism, which permits independent regulation of odorant detection from survival signaling, may be advantageous if not diminished by repeated or prolonged odor exposure.

     

Altered odor‐induced expression of c‐fos and arg 3.1 immediate early genes in the olfactory system after familiarization with an odor

In adult rats, repeated exposure to an odorant, in absence of any experimentally delivered reinforcement, leads to a drastic decrease in mitral/tufted (M/T) cell responsiveness, not only for the familiar odor but also for other novel odors. In the present study, using two different and complementary in situ hybridization methods, we analyzed the effect of familiarization with an odorant on c‐fos and arg 3.1 mRNA expression levels, and we examined the odor specificity of this effect. Odor exposure induces a specific increase in c‐fos and arg 3.1 expression in some particular olfactory bulb quadrants. Previous familiarization with the test odor results in a decreased expression of both IEGs in these quadrants, leading to the alteration of the odor‐specific pattern of c‐fos and arg 3.1 expression. In contrast, this odor‐specific pattern is not affected when different odors are used for familiarization and test. Similarly, an odor‐specific familiarization effect leading to a reduced c‐fos and arg 3.1 expression was also detected in the cingulate cortex and in the anterior piriform cortex. These results support our hypothesis that the decrease in M/T cell responsiveness following a preceding familiarization with an odorant may be related to a particular form of synaptic plasticity involving changes at the genomic level, and reveals further insight in olfactory information processing and the cellular mechanisms underlying familiarization in the olfactory system. © 2002 Wiley Periodicals, Inc. J Neurobiol 52: 61–72, 2002     

N-formyl peptide receptor phosphorylation domains differentially regulate arrestin and agonist affinity

Arrestins regulate the signaling and endocytosis of many G protein-coupled receptors (GPCRs). It has been suggested that the functions of arrestins are dependent upon both the number and pattern of phosphorylation sites present in an activated GPCR. However, little is currently known about the relationships between the sites of receptor phosphorylation, the resulting affinities of arrestin binding, and the ensuing mechanisms of receptor regulation for any given GPCR. To investigate these interactions, we used an active truncated mutant of arrestin (amino acids 1-382) and phosphorylation-deficient mutants of the N-formyl peptide receptor (FPR). In contrast to results with wild type arrestins, the truncated arrestin-2 protein bound to the unphosphorylated wild type FPR, although with lower affinity and a low affinity for the agonist as revealed by competition studies with heterotrimeric G proteins. Using FPR mutants, we further demonstrated that the phosphorylation status of serines and threonines between residues 328-332 is a key determinant that regulates the affinity of the FPR for arrestins. Furthermore, we found that the phosphorylation status of serine and threonine residues between amino acids 334 and 339 regulates the affinity of the receptor for agonist when arrestin is bound. These results suggest that the agonist affinity state of the receptor is principally regulated by phosphorylation at specific sites and is not simply a consequence of arrestin binding as has previously been proposed. Furthermore, this is the first demonstration that agonist affinity of a GPCR and the affinity of arrestin binding to the phosphorylated receptor are regulated by distinct receptor phosphodomains.     

Regulation of N-Formyl Peptide Receptor Signaling and Trafficking by Arrestin-Src Kinase Interaction

Arrestins were originally described as proteins recruited to ligand-activated, phosphorylated G protein-coupled receptors (GPCRs) to attenuate G protein-mediated signaling. It was later revealed that arrestins also mediate GPCR internalization and recruit a number of signaling proteins including, but not limited to, Src family kinases, ERK1/2, and JNK3. GPCR-arrestin binding and trafficking control the spatial and temporal activity of these multi-protein complexes. In previous reports, we concluded that N-formyl peptide receptor (FPR)-mediated apoptosis, which occurs upon receptor stimulation in the absence of arrestins, is associated with FPR accumulation in perinuclear recycling endosomes. Under these conditions, inhibition of Src kinase and ERK1/2 prevented FPR-mediated apoptosis. To better understand the role of Src kinase in this process, in the current study we employed a previously described arrestin-2 (arr2) mutant deficient in Src kinase binding (arr2-P91G/P121E). Unlike wild type arrestin, arr2-P91G/P121E did not inhibit FPR-mediated apoptosis, suggesting that Src binding to arrestin-2 prevents apoptotic signaling. However, in cells expressing this mutant, FPR-mediated apoptosis was still blocked by inhibition of Src kinase activity, suggesting that activation of Src independent of arrestin-2 binding is involved in FPR-mediated apoptosis. Finally, while Src kinase inhibition prevented FPR-mediated-apoptosis in the presence of arr2-P91G/P121E, it did not prevent FPR-arr2-P91G/P121E accumulation in the perinuclear recycling endosome. On the contrary, inhibition of Src kinase activity mediated the accumulation of activated FPR-wild type arrestin-2 in recycling endosomes without initiating FPR-mediated apoptosis. Based on these observations, we conclude that Src kinase has two independent roles following FPR activation that regulate both FPR-arrestin-2 signaling and trafficking.     

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-beta receptors. These activities indicate broad roles in both physiology and development for nonvisual arrestins. Drosophila melanogaster 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, we rescued krz(1) lethality. The resulting adults lacked detectable levels of krz in the olfactory system. The rescued krz(1) homozygotes have an incompletely penetrant antennal structural defect that was completely rescued by the neural expression of a krz cDNA. The krz(1) 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 krz(1) antenna indicate that nonvisual arrestins are required for the early odor-induced signaling events within the ORNs.     

Human alpha‐ and beta‐NRXN1 isoforms rescue behavioral impairments of Caenorhabditis elegans neurexin‐deficient mutants

Neurexins are cell adhesion proteins that interact with neuroligin and other ligands at the synapse. In humans, mutations in neurexin or neuroligin genes have been associated with autism and other mental disorders. The human neurexin and neuroligin genes are orthologous to the Caenorhabditis elegans genes nrx‐1 and nlg‐1, respectively. Here we show that nrx‐1‐deficient mutants are defective in exploratory capacity, sinusoidal postural movements and gentle touch response. Interestingly, the exploratory behavioral phenotype observed in nrx‐1 mutants was markedly different to nlg‐1‐deficient mutants; thus, while the former had a ‘hyper‐reversal’ phenotype increasing the number of changes of direction with respect to the wild‐type strain, the nlg‐1 mutants presented a ‘hypo‐reversal’ phenotype. On the other hand, the nrx‐1‐ and nlg‐1‐defective mutants showed similar abnormal sinusoidal postural movement phenotypes. The response of these mutant strains to aldicarb (acetylcholinesterase inhibitor), levamisole (ACh agonist) and pentylenetetrazole [gamma‐aminobutyric (GABA) receptor antagonist], suggested that the varying behavioral phenotypes were caused by defects in ACh and/or GABA inputs. The defective behavioral phenotypes of nrx‐1‐deficient mutants were rescued in transgenic strains expressing either human alpha‐ or beta‐NRXN‐1 isoforms under the worm nrx‐1 promoter. A previous report had shown that human and rat neuroligins were functional in C. elegans. Together, these results suggest that the functional mechanism underpinning both neuroligin and neurexin in the nematode are comparable to human. In this sense the nematode might constitute a simple in vivo model for understanding basic mechanisms involved in neurological diseases for which neuroligin and neurexin are implicated in having a role.     

Caenorhabditus elegans arrestin regulates neural G protein signaling and olfactory adaptation and recovery

Although regulation of G protein-coupled receptor signaling by receptor kinases and arrestins is a well established biochemical process, the physiological significance of such regulation remains poorly understood. To better understand the in vivo consequences of arrestin function, we have examined the function of the sole arrestin in Caenorhabditis elegans (ARR-1). ARR-1 is primarily expressed in the nervous system, including the HSN neuron and various chemosensory neurons involved in detecting soluble and volatile odorants. arr-1 null mutants exhibit normal chemotaxis but have significant defects in olfactory adaptation and recovery to volatile odorants. In contrast, adaptation is enhanced in animals overexpressing ARR-1. Both the adaptation and recovery defects of arr-1 mutants are rescued by transgenic expression of wild-type ARR-1, whereas expression of a C-terminally truncated ARR-1 effectively rescues only the adaptation defect. A potential mechanistic basis for these findings is revealed by in vitro studies demonstrating that wild-type ARR-1 binds proteins of the endocytic machinery and promotes receptor endocytosis, whereas C-terminally truncated ARR-1 does not. These results demonstrate that ARR-1 functions to regulate chemosensory signaling, enabling organisms to adapt to a variety of environmental cues, and provide an in vivo link between arrestin, receptor endocytosis, and temporal recovery from adaptation.     

V1b vasopressin receptor trafficking and signaling: Role of arrestins,G proteins and Src kinase

The signaling pathway of G protein‐coupled receptors is strongly linked to their trafficking profile. Little is known about the molecular mechanisms involved in the vasopressin receptor V_1b subtype (V_1bR) trafficking and its impact on receptor signaling and regulation. For this purpose, we investigated the role of β‐arrestins in receptor desensitization, internalization and recycling and attempted to dissect the V_1bR‐mediated MAP kinase pathway. Using MEF cells Knocked‐out for β‐arrestins 1 and 2, we demonstrated that both β‐arrestins 1 and 2 play a fundamental role in internalization and recycling of V_1bR with a rapid and transient V_1bR‐β‐arrestin interaction in contrast to a slow and long‐lasting β‐arrestin recruitment of the V₂ vasopressin receptor subtype (V₂R). Using V_1bR‐V₂R chimeras and V_1bR C‐terminus truncations, we demonstrated the critical role of the V_1bR C‐terminus in its interaction with β‐arrestins thereby regulating the receptor internalization and recycling kinetics in a phosphorylation‐independent manner. In parallel, V_1bR MAP kinase activation was dependent on arrestins and Src‐kinase but independent on G proteins. Interestingly, Src interacted with hV_1bR at basal state and dissociated when receptor internalization occurred. Altogether, our data describe for the first time the trafficking profile and MAP kinase pathway of V_1bR involving both arrestins and Src kinase family.      

Evidence for speciation underground in diving beetles (Dytiscidae) from a subterranean archipelago

Most subterranean animals are assumed to have evolved from surface ancestors following colonization of a cave system; however, very few studies have raised the possibility of “subterranean speciation” in underground habitats (i.e., obligate cave‐dwelling organisms [troglobionts] descended from troglobiotic ancestors). Numerous endemic subterranean diving beetle species from spatially discrete calcrete aquifers in Western Australia (stygobionts) have evolved independently from surface ancestors; however, several cases of sympatric sister species raise the possibility of subterranean speciation. We tested this hypothesis using vision (phototransduction) genes that are evolving under neutral processes in subterranean species and purifying selection in surface species. Using sequence data from 32 subterranean and five surface species in the genus Paroster (Dytiscidae), we identified deleterious mutations in long wavelength opsin (lwop), arrestin 1 (arr1), and arrestin 2 (arr2) shared by a sympatric sister‐species triplet, arr1 shared by a sympatric sister‐species pair, and lwop and arr2 shared among closely related species in adjacent calcrete aquifers. In all cases, a common ancestor possessed the function‐altering mutations, implying they were already adapted to aphotic environments. Our study represents one of the first confirmed cases of subterranean speciation in cave insects. The assessment of genes undergoing pseudogenization provides a novel way of testing modes of speciation and the history of diversification in blind cave animals.     

Intranasal Odorant Concentrations in Relation to Sniff Behavior

Knowledge on how odorants are transported through the nasal cavity to the olfactory epithelium is limited. One facet of this is how the sniffing behavior affects the abundance of odorants transferred to the olfactory cleft and in turn influences odor perception. A novel system that couples an online mass spectrometer with an odorant pulse delivery olfactometer was employed to characterize intranasal odorant concentrations of butane‐2,3‐dione (or butanedione, commonly known as diacetyl) at the interior naris and the olfactory cleft. Volunteers (n=12) were asked to perform different modes of sniffing in relation to the sniff intensity that were categorized as ‘normal’, ‘rapid’ and ‘forced’. The highest concentrations of butanedione at both positions in the nose were observed during normal sniffing, with the lowest concentrations correlating with periods of forced sniffs. This corresponded to the panelists' ratings that normal sniffing elicited the highest odor intensities. These feasibility assessments pave the way for more in‐depth analyses with a variety of odorants of different chemical classes at various intranasal positions, to investigate the passage and uptake of odorants within the nasal cavity.     

Stress physiology functions of the Arabidopsis histidine kinase cytokinin receptors

Cytokinin signaling has complex effects on abiotic stress responses that remain to be fully elucidated. The Arabidopsis histidine kinases (AHKs), AHK2, AHK3 and CRE1 (cytokinin response1/AHK4) are the principle cytokinin receptors of Arabidopsis. Using a set of ahk mutants, we found dramatic differences in response to low water potential and salt stress among the AHKs. ahk3‐3 mutants had increased root elongation after transfer to low water potential media. Conversely ahk2‐2 was hypersensitive to salt stress in terms of root growth and fresh weight and accumulated higher than wild‐type levels of proline specifically under salt stress. Strongly reduced proline accumulation in ahk double mutants after low water potential treatment indicated a more general role of cytokinin signaling in proline metabolism. Reduced P5CS1 (Δ¹‐pyrroline‐5‐carboxylate synthetase1) gene expression may have contributed to this reduced proline accumulation. Low water potential phenotypes of ahk mutants were not caused by altered abscisic acid (ABA) accumulation as all ahk mutants had wild‐type ABA levels, despite the observation that ahk double mutants had reduced NCED3 (9‐cis‐epoxycartenoid dioxygenase3) expression when exposed to low water potential. No difference in osmoregulatory solute accumulation was detected in any of the ahk mutants indicating that they do not affect drought responsive osmotic adjustment. Overall, our examination of ahk mutants found specific phenotypes associated with AHK2 and AHK3 as well as a general function of cytokinin signaling in proline accumulation and low water potential induction of P5CS1 and NCED3 expression. These results show the stress physiology function of AHKs at a new level of detail.     

Depletion of β‐arrestin2 in hepatic stellate cells reduces cell proliferation via ERK pathway

β‐Arrestins are multifunctional adaptor proteins. Recently, some new roles of β‐arrestins in regulating intracellular signaling networks have been discovered, which regulate cell growth, proliferation, and apoptosis. Though, the role of β‐arrestins expression in the pathology of hepatic fibrosis remains unclear. In this study, the possible relationship between the expression of β‐arrestins with the experimental hepatic fibrosis and the proliferation of hepatic stellate cells (HSCs) were investigated. Porcine serum induced liver fibrosis was established in this study. At five time points, the dynamic expression of β‐arrestin1, β‐arrestin2, and α‐smooth muscle actin (α‐SMA) in rat liver tissues, was measured by immunohistochemical staining, double immunofluorescent staining, and Western blotting. This study showed that aggravation of hepatic fibrosis with gradually increasing expression of β‐arrestin2 in the hepatic tissues, but not β‐arrestin1. Further, as hepatic fibrosis worsens, β‐arrestin2‐expressing activated HSCs accounts for an increasingly larger percentage of all activated HSCs. And the expression of β‐arrestin2 had a significant positive correlation with the expression of α‐SMA, an activated HSCs marker. In vitro studies, the dynamic expression of β‐arrestin1 and β‐arrestin2 in platelet derived growth factor‐BB (PDGF‐BB) stimulated HSCs was assessed by Western blotting. The expression of β‐arrestin2 was remarkably increased in PDGF‐BB stimulated HSCs. Furthermore, the small interfering RNA (siRNA) technique was used to explore the effect of β‐arrestins on the proliferation of HSCs and the activation of ERK1/2. Transfection of siRNA targeting β‐arrestin2 mRNA (siβ‐arrestin2) into HSCs led to a 68% and 70% reduction of β‐arrestin2 mRNA and protein expression, respectively. siβ‐arrestin2 abolished the effect of PDGF‐BB on the proliferation of HSCs. In addition, siβ‐arrestin2 exerted the inhibition of the activation of ERK1/2 in HSCs. The present study provided strong evidence for the participation of the β‐arrestin2 in the pathogenesis of hepatic fibrosis. The β‐arrestin2 depletion diminishes HSCs ERK1/2 signaling and proliferation stimulated by PDGF‐BB. Selective targeting of β‐arrestin2 inhibitors to HSCs might present as a novel strategy for the treatment of hepatic fibrosis. J. Cell. Biochem. 114: 1153–1162, 2013. © 2012 Wiley Periodicals, Inc.     

Cone arrestin binding to JNK3 and Mdm2: conformational preference and localization of interaction sites

Arrestins are multi-functional regulators of G protein-coupled receptors. Receptor-bound arrestins interact with >30 remarkably diverse proteins and redirect the signaling to G protein-independent pathways. The functions of free arrestins are poorly understood, and the interaction sites of the non-receptor arrestin partners are largely unknown. In this study, we show that cone arrestin, the least studied member of the family, binds c-Jun N-terminal kinase (JNK3) and Mdm2 and regulates their subcellular distribution. Using arrestin mutants with increased or reduced structural flexibility, we demonstrate that arrestin in all conformations binds JNK3 comparably, whereas Mdm2 preferentially binds cone arrestin 'frozen' in the basal state. To localize the interaction sites, we expressed separate N- and C-domains of cone and rod arrestins and found that individual domains bind JNK3 and remove it from the nucleus as efficiently as full-length proteins. Thus, the arrestin binding site for JNK3 includes elements in both domains with the affinity of partial sites on individual domains sufficient for JNK3 relocalization. N-domain of rod arrestin binds Mdm2, which localizes its main interaction site to this region. Comparable binding of JNK3 and Mdm2 to four arrestin subtypes allowed us to identify conserved residues likely involved in these interactions.     

A cyclic nucleotide-gated channel in the brain regulates olfactory modulation through neuropeptide F in fruit fly Drosophila melanogaster

Olfactory sensing and its modulation are important for the insects in recognizing diverse odors from the environment and in making correct decisions to survive. Identifying new genes involved in olfactory modulation and unveiling their mechanisms may lead us to understand decision making processes in the central nervous system. Here, we report a novel olfactory function of the cyclic nucleotide-gated (CNG) channel CG42260 in modulating ab3A olfactory sensory neurons, which specifically respond to food-derived odors in fruit fly Drosophila melanogaster. We found that two independent CG42260 mutants show reduced responses in the ab3A neurons. Unlike mammalian CNGs, CG42260 is not expressed in the odorant sensory neurons but broadly in the central nervous system including neuropeptide-producing cells. By using molecular genetic tools, we identified CG42260 expression in one pair of neuropeptide F (NPF) positive L1-l cells known to modulate food odor responsiveness. Knockdown of CG42260 in the NPF neurons reduced production of NPF in Ll-1 cells, which in turn, led to reduction of neuronal responses of the ab3A neurons. Our findings show the novel biological function of CG42260 in modulating olfactory responses to food odor through NPF.     

Ubiquitin ligase parkin promotes Mdm2-arrestin interaction but inhibits arrestin ubiquitination

Numerous mutations in E3 ubiquitin ligase parkin were shown to associate with familial Parkinson's disease. Here we show that parkin binds arrestins, versatile regulators of cell signaling. Arrestin-parkin interaction was demonstrated by coimmunoprecipitation of endogenous proteins from brain tissue and shown to be direct using purified proteins. Parkin binding enhances arrestin interactions with another E3 ubiquitin ligase, Mdm2, apparently by shifting arrestin conformational equilibrium to the basal state preferred by Mdm2. Although Mdm2 was reported to ubiquitinate arrestins, parkin-dependent increase in Mdm2 binding dramatically reduces the ubiquitination of both nonvisual arrestins, basal and stimulated by receptor activation, without affecting receptor internalization. Several disease-associated parkin mutations differentially affect the stimulation of Mdm2 binding. All parkin mutants tested effectively suppress arrestin ubiquitination, suggesting that bound parkin shields arrestin lysines targeted by Mdm2. Parkin binding to arrestins along with its effects on arrestin interaction with Mdm2 and ubiquitination is a novel function of this protein with implications for Parkinson's disease pathology.