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1.
PLoS One ; 19(6): e0303882, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38848405

RESUMO

Activated GPCRs are phosphorylated and internalized mostly via clathrin-mediated endocytosis (CME), which are then sorted for recycling or degradation. We investigated how differential activation of the same GPCR affects its endocytic trafficking in vivo using rhodopsin as a model in pupal photoreceptors of flies expressing mCherry-tagged rhodopsin 1 (Rh1-mC) or GFP-tagged arrestin 1 (Arr1-GFP). Upon blue light stimulation, activated Rh1 recruited Arr1-GFP to the rhabdomere, which became co-internalized and accumulated in cytoplasmic vesicles of photoreceptors. This internalization was eliminated in shits1 mutants affecting dynamin. Moreover, it was blocked by either rdgA or rdgB mutations affecting the PIP2 biosynthesis. Together, the blue light-initiated internalization of Rh1 and Arr1 belongs to CME. Green light stimulation also triggered the internalization and accumulation of activated Rh1-mC in the cytoplasm but with faster kinetics. Importantly, Arr1-GFP was also recruited to the rhabdomere but not co-internalized with Rh1-mC. This endocytosis was not affected in shits1 nor rdgA mutants, indicating it is not CME. We explored the fate of internalized Rh1-mC following CME and observed it remained in cytoplasmic vesicles following 30 min of dark adaptation. In contrast, in the non-CME Rh1-mC appeared readily recycled back to the rhabdomere within five min of dark treatment. This faster recycling may be regulated by rhodopsin phosphatase, RdgC. Together, we demonstrate two distinct endocytic and recycling mechanisms of Rh1 via two light stimulations. It appears that each stimulation triggers a distinct conformation leading to different phosphorylation patterns of Rh1 capable of recruiting Arr1 to rhabdomeres. However, a more stable interaction leads to the co-internalization of Arr1 that orchestrates CME. A stronger Arr1 association appears to impede the recycling of the phosphorylated Rh1 by preventing the recruitment of RdgC. We conclude that conformations of activated rhodopsin determine the downstream outputs upon phosphorylation that confers differential protein-protein interactions.


Assuntos
Endocitose , Rodopsina , Rodopsina/metabolismo , Rodopsina/genética , Animais , Fosforilação , Transporte Proteico , Luz , Mutação , Células Fotorreceptoras de Invertebrados/metabolismo , Drosophila melanogaster/metabolismo , Clatrina/metabolismo
2.
J Biol Chem ; 299(6): 104822, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37201584

RESUMO

Pkc53E is the second conventional protein kinase C (PKC) gene expressed in Drosophila photoreceptors; it encodes at least six transcripts generating four distinct protein isoforms including Pkc53E-B whose mRNA is preferentially expressed in photoreceptors. By characterizing transgenic lines expressing Pkc53E-B-GFP, we show Pkc53E-B is localized in the cytosol and rhabdomeres of photoreceptors, and the rhabdomeric localization appears dependent on the diurnal rhythm. A loss of function of pkc53E-B leads to light-dependent retinal degeneration. Interestingly, the knockdown of pkc53E also impacted the actin cytoskeleton of rhabdomeres in a light-independent manner. Here the Actin-GFP reporter is mislocalized and accumulated at the base of the rhabdomere, suggesting that Pkc53E regulates depolymerization of the actin microfilament. We explored the light-dependent regulation of Pkc53E and demonstrated that activation of Pkc53 E can be independent of the phospholipase C PLCß4/NorpA as degeneration of norpAP24 photoreceptors was enhanced by a reduced Pkc53E activity. We further show that the activation of Pkc53E may involve the activation of Plc21C by Gqα. Taken together, Pkc53E-B appears to exert both constitutive and light-regulated activity to promote the maintenance of photoreceptors possibly by regulating the actin cytoskeleton.


Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Proteína Quinase C , Animais , Citoesqueleto de Actina/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Fosfolipases/metabolismo , Células Fotorreceptoras de Invertebrados/metabolismo , Proteína Quinase C/genética , Proteína Quinase C/metabolismo
3.
Fly (Austin) ; 15(1): 8-27, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-33200658

RESUMO

Unregulated Ca2+ influx affects intracellular Ca2+ homoeostasis, which may lead to neuronal death. In Drosophila, following the activation of rhodopsin the TRP Ca2+ channel is open to mediate the light-dependent depolarization. A constitutively active TRP channel triggers the degeneration of TrpP365 /+ photoreceptors. To explore retinal degeneration, we employed a multidisciplinary approach including live imaging using GFP tagged actin and arrestin 2. Importantly, we demonstrate that the major rhodopsin (Rh1) was greatly reduced before the onset of rhabdomere degeneration; a great reduction of Rh1 affects the maintenance of rhabdomere leading to degeneration of photoreceptors. TrpP365 /+ also led to the up-regulation of CaMKII, which is beneficial as suppression of CaMKII accelerated retinal degeneration. We explored the regulation of TRP by investigating the genetic interaction between TrpP365 /+ and mutants affecting the turnover of diacylglycerol (DAG). We show a loss of phospholipase C in norpAP24 exhibited a great reduction of the DAG content delayed degeneration of TrpP365 /+ photoreceptors. In contrast, knockdown or mutations in DAG lipase (InaE) that is accompanied by slightly reduced levels of most DAG but an increased level of DAG 34:1, exacerbated retinal degeneration of TrpP365 /+. Together, our findings support the notion that DAG plays a role in regulating TRP. Interestingly, DAG lipase is likely required during photoreceptor development as TrpP365 /+; inaEN125 double mutants contained severely degenerated rhabdomeres.


Assuntos
Canais de Cálcio/metabolismo , Drosophila/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Actinas , Animais , Animais Geneticamente Modificados , Arrestinas/genética , Arrestinas/metabolismo , Canais de Cálcio/genética , Diglicerídeos/metabolismo , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Proteínas de Fluorescência Verde , Luz , Mutação , Fosfolipase C beta/genética , Fosfolipase C beta/metabolismo , Pupa , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rodopsina , Canais de Potencial de Receptor Transitório/genética
4.
J Biol Chem ; 289(26): 18526-34, 2014 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-24838243

RESUMO

Arrestin regulates many facets of G-protein coupled receptor signaling. In Drosophila, Arrestin 1 (Arr1) is expressed at a lower level than Arrestin 2 (Arr2), and the role of Arr1 in visual physiology is less understood. Here we generated transgenic flies expressing enhanced green fluorescent protein tagged Arr1 (Arr1-eGFP) and explored its trafficking in live photoreceptors. We show that Arr1-eGFP is localized in the cytoplasm and displays light-dependent translocation to the rhabdomere possibly by interacting with photoactivated rhodopsin 1 (Rh1*). In the adult, translocation of Arr1-eGFP occurs with slower kinetics when compared with that of Arr2-eGFP. This slower kinetic activity may be attributable to a reduced level of phosphorylated Rh1*. Indeed, a reduced level of phosphorylated Rh1* recruits a lower level of Arr1-eGFP to rhabdomeres. To investigate whether Arr1 is required for the deactivation of phosphorylated Rh1*, we show that in flies with reduced Arr1 prolonged depolarizing afterpotential can be triggered with fewer light pulses, indicating that inactivation of phosphorylated Rh1* is compromised when the Arr1 level is reduced. Consistently, Arr1 is no longer required for deactivation of Rh1 in flies expressing phosphorylation-deficient Rh1. Previously it was reported that Arr1 displays light-dependent internalization. Unexpectedly, in adult photoreceptors we failed to observe endocytosis of Arr1-eGFP. In contrast, we show that in pupal photoreceptors Arr1-eGFP becomes internalized and sequestered in vesicles within the cytoplasm. Taken together, we propose that Arr1 plays distinct roles during development and adulthood. Arr1 orchestrates the recycling of phosphorylated Rh1* in pupae whereas it regulates the deactivation in adult.


Assuntos
Arrestinas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Células Fotorreceptoras/metabolismo , Pupa/crescimento & desenvolvimento , Animais , Arrestinas/genética , Drosophila/genética , Drosophila/crescimento & desenvolvimento , Proteínas de Drosophila/genética , Fosforilação , Transporte Proteico , Pupa/genética , Pupa/metabolismo , Rodopsina/genética , Rodopsina/metabolismo
5.
J Neurosci ; 32(31): 10758-66, 2012 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-22855823

RESUMO

Arrestins belong to a family of multifunctional adaptor proteins that regulate internalization of diverse receptors including G-protein-coupled receptors (GPCRs). Defects associated with endocytosis of GPCRs have been linked to human diseases. We used enhanced green fluorescent protein-tagged arrestin 2 (Arr2) to monitor the turnover of the major rhodopsin (Rh1) in live Drosophila. We demonstrate that during degeneration of norpA(P24) photoreceptors the loss of Rh1 is parallel to the disappearance of rhabdomeres, the specialized visual organelle that houses Rh1. The cause of degeneration in norpA(P24) is the failure to activate CaMKII (Ca(2+)/calmodulin-dependent protein kinase II) and retinal degeneration C (RDGC) because of a loss of light-dependent Ca(2+) entry. A lack of activation in CaMKII, which phosphorylates Arr2, leads to hypophosphorylated Arr2, while a lack of activation of RDGC, which dephosphorylates Rh1, results in hyperphosphorylated Rh1. We investigated how reversible phosphorylation of Rh1 and Arr2 contributes to photoreceptor degeneration. To uncover the consequence underlying a lack of CaMKII activation, we characterized ala(1) flies in which CaMKII was suppressed by an inhibitory peptide, and showed that morphology of rhabdomeres was not affected. In contrast, we found that expression of phosphorylation-deficient Rh1s, which either lack the C terminus or contain Ala substitution in the phosphorylation sites, was able to prevent degeneration of norpA(P24) photoreceptors. This suppression is not due to a loss of Arr2 interaction. Importantly, co-expression of these modified Rh1s offered protective effects, which greatly delayed photoreceptor degeneration. Together, we conclude that phosphorylation of Rh1 is the major determinant that orchestrates its internalization leading to retinal degeneration.


Assuntos
Arrestinas/metabolismo , Proteínas de Drosophila/metabolismo , Degeneração Retiniana/metabolismo , Rodopsina/metabolismo , Animais , Animais Geneticamente Modificados , Arrestinas/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Modelos Animais de Doenças , Drosophila , Proteínas de Drosophila/genética , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/genética , Proteínas de Fluorescência Verde/genética , Humanos , Mutação/genética , Fosfolipase C beta/genética , Fosforilação/genética , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Degeneração Retiniana/genética , Degeneração Retiniana/patologia , Rodopsina/genética , Isótopos de Enxofre/farmacologia
6.
Fly (Austin) ; 5(4): 356-68, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21897116

RESUMO

Inherited retinal degeneration in Drosophila has been explored for insights into similar processes in humans. Based on the mechanisms, I divide these mutations in Drosophila into three classes. The first consists of genes that control the specialization of photoreceptor cells including the morphogenesis of visual organelles  (rhabdomeres) that house the visual signaling proteins. The second class contains genes that regulate the activity or level of the major rhodopsin, Rh1, which is the light sensor and also provides a structural role for the maintenance of rhabdomeres. Some mutations in Rh1 (NinaE) are dominant due to constitutive activity or folding defects, like autosomal dominant retinitis pigmentosa (ADRP) in humans. The third class consists of genes that control the Ca ( 2+) influx directly or indirectly by promoting the turnover of the second messenger and regeneration of PIP 2, or mediate the Ca ( 2+) -dependent regulation of the visual response. These gene products are critical for the increase in cytosolic Ca ( 2+ ) following light stimulation to initiate negative regulatory events. Here I will focus on the signaling mechanisms underlying the degeneration in norpA, and in ADRP-type NinaE mutants that produce misfolded Rh1. Accumulation of misfolded Rh1 in the ER triggers the unfolded protein response (UPR), while endosomal accumulation of activated Rh1 may initiate autophagy in norpA. Both autophagy and the UPR are beneficial for relieving defective endosomal trafficking and the ER stress, respectively. However, when photoreceptors fail to cope with the persistence of these stresses, a cell death program is activated leading to retinal degeneration.


Assuntos
Drosophila/genética , Células Fotorreceptoras de Invertebrados/citologia , Degeneração Retiniana/genética , Animais , Autofagia , Cálcio/metabolismo , Modelos Animais de Doenças , Drosophila/citologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Humanos , Modelos Biológicos , Fosfolipase C beta/genética , Fosfolipase C beta/metabolismo , Fosfolipase C beta/fisiologia , Células Fotorreceptoras de Invertebrados/metabolismo , Células Fotorreceptoras de Invertebrados/patologia , Dobramento de Proteína , Degeneração Retiniana/patologia , Rodopsina/genética , Rodopsina/metabolismo , Rodopsina/fisiologia , Transdução de Sinais
7.
J Biol Chem ; 284(17): 11100-9, 2009 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-19254957

RESUMO

Ca(2+) modulates the visual response in both vertebrates and invertebrates. In Drosophila photoreceptors, an increase of cytoplasmic Ca(2+) mimics light adaptation. Little is known regarding the mechanism, however. We explored the role of the sole Drosophila Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) to mediate light adaptation. CaMKII has been implicated in the phosphorylation of arrestin 2 (Arr2). However, the functional significance of Arr2 phosphorylation remains debatable. We identified retinal CaMKII by anti-CaMKII antibodies and by its Ca(2+)-dependent autophosphorylation. Moreover, we show that phosphorylation of CaMKII is greatly enhanced by okadaic acid, and indeed, purified PP2A catalyzes the dephosphorylation of CaMKII. Significantly, we demonstrate that anti-CaMKII antibodies co-immunoprecipitate, and CaMKII fusion proteins pull down the catalytic subunit of PP2A from fly extracts, indicating that PP2A interacts with CaMKII to form a protein complex. To investigate the function of CaMKII in photoreceptors, we show that suppression of CaMKII in transgenic flies affects light adaptation and increases prolonged depolarizing afterpotential amplitude, whereas a reduced PP2A activity brings about reduced prolonged depolarizing afterpotential amplitude. Taken together, we conclude that CaMKII is involved in the negative regulation of the visual response affecting light adaptation, possibly by catalyzing phosphorylation of Arr2. Moreover, the CaMKII activity appears tightly regulated by the co-localized PP2A.


Assuntos
Arrestinas/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/fisiologia , Cálcio/metabolismo , Proteínas de Drosophila/metabolismo , Células Fotorreceptoras de Invertebrados/metabolismo , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Calmodulina/química , Catálise , Domínio Catalítico , Citosol/metabolismo , Drosophila , Eletrorretinografia/métodos , Heterozigoto , Fenótipo , Fosforilação , Estrutura Terciária de Proteína
8.
J Neurosci ; 28(6): 1444-51, 2008 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-18256265

RESUMO

Drosophila visual signaling, a G-protein-coupled phospholipase Cbeta (PLCbeta)-mediated mechanism, is regulated by eye-protein kinase C (PKC) that promotes light adaptation and fast deactivation, most likely via phosphorylation of inactivation no afterpotential D (INAD) and TRP (transient receptor potential). To reveal the critical phosphatases that dephosphorylate INAD, we used several biochemical analyses and identified protein phosphatase 2A (PP2A) as a candidate. Importantly, the catalytic subunit of PP2A, microtubule star (MTS), is copurified with INAD, and an elevated phosphorylation of INAD by eye-PKC was observed in three mts heterozygotes. To explore whether PP2A (MTS) regulates dephosphorylation of INAD by counteracting eye-PKC [INAC (inactivation no afterpotential C] in vivo, we performed ERG recordings. We discovered that inaC(P209) was semidominant, because inaC(P209) heterozygotes displayed abnormal light adaptation and slow deactivation. Interestingly, the deactivation defect of inaC(P209) heterozygotes was rescued by the mts(XE2258) heterozygous background. In contrast, mts(XE2258) failed to modify the severe deactivation of norpA(P16), indicating that MTS does not modulate NORPA (no receptor potential A) (PLCbeta). Together, our results strongly indicate that dephosphorylation of INAD is catalyzed by PP2A, and a reduction of PP2A can compensate for a partial loss of function in eye-PKC, restoring the fast deactivation kinetics in vivo. We thus propose that the fast deactivation of the visual response is modulated in part by the phosphorylation of INAD.


Assuntos
Proteínas de Drosophila/fisiologia , Células Fotorreceptoras de Invertebrados/fisiologia , Proteína Fosfatase 2/fisiologia , Transdução de Sinais/fisiologia , Percepção Visual/fisiologia , Potenciais de Ação/fisiologia , Adaptação Ocular/fisiologia , Sequência de Aminoácidos , Animais , Catálise , Drosophila , Proteínas de Drosophila/metabolismo , Proteínas do Olho/metabolismo , Proteínas do Olho/fisiologia , Feminino , Masculino , Dados de Sequência Molecular , Fosforilação
9.
J Neurochem ; 104(6): 1526-35, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18036153

RESUMO

Drosophila transient-receptor-potential (TRP) is a Ca2+ channel responsible for the light-dependent depolarization of photoreceptors. TRP is anchored to a macromolecular complex by tethering to inactivation-no-afterpotential D (INAD). We previously reported that INAD associated with the carboxyl tail of TRP via its third post-synaptic density protein 95, discs-large, zonular occludens-1 domain. In this paper, we further explored the molecular basis of the INAD interaction and demonstrated the requirement of the last 14 residues of TRP, with the critical contribution of Gly1262, Val1266, Trp1274, and Leu1275. We also revealed by pull-down assays that the last 14 residues of TRP comprised the minimal sequence that competes with the endogenous TRP from fly extracts, leading to the co-purification of a partial INAD complex containing INAD, no-receptor-potential A, and eye-protein kinase C (PKC). Eye-PKC is critical for the negative regulation of the visual signaling and was shown to phosphorylate TRP in vivo. To uncover the substrates of eye-PKC in the INAD complex, we designed a complex-dependent eye-PKC assay, which utilized endogenous INAD complexes isolated from flies. We demonstrate that activated eye-PKC phosphorylates INAD, TRP but not no-receptor-potential A. Moreover, phosphorylation of TRP is dependent on the presence of both eye-PKC and INAD. Together, these findings indicate that stable kinase-containing protein complexes may be isolated by pull-down assays, and used in this modified kinase assay to investigate phosphorylation of the proteins in the complex. We conclude that TRP associates with INAD via its last 14 residues to facilitate its regulation by eye-PKC that fine-tunes the visual signaling.


Assuntos
Proteínas de Drosophila/metabolismo , Proteínas do Olho/metabolismo , Células Fotorreceptoras de Invertebrados/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Visão Ocular/fisiologia , Animais , Drosophila , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas do Olho/química , Proteínas do Olho/genética , Glicina/metabolismo , Leucina/metabolismo , Substâncias Macromoleculares/metabolismo , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/metabolismo , Fosfolipase C beta/metabolismo , Fosforilação , Proteína Quinase C/metabolismo , Estrutura Terciária de Proteína , Serina/metabolismo , Canais de Potencial de Receptor Transitório/química , Canais de Potencial de Receptor Transitório/genética , Triptofano/metabolismo , Canais de Ânion Dependentes de Voltagem
10.
J Neurosci ; 26(33): 8570-7, 2006 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-16914683

RESUMO

Drosophila visual signaling is one of the fastest G-protein-coupled transduction cascades, because effector and modulatory proteins are organized into a macromolecular complex ("transducisome"). Assembly of the complex is orchestrated by inactivation no afterpotential D (INAD), which colocalizes the transient receptor potential (TRP) Ca2+ channel, phospholipase Cbeta, and eye protein kinase C (eye-PKC), for more efficient signal transduction. Eye-PKC is critical for deactivation of vision. Moreover, deactivation is regulated by the interaction between INAD and TRP, because abrogation of this interaction in InaD(p215) results in slow deactivation similar to that of inaC(p209) lacking eye-PKC. To elucidate the mechanisms whereby eye-PKC modulates deactivation, here we demonstrate that eye-PKC, via tethering to INAD, phosphorylates TRP in vitro. We reveal that Ser982 of TRP is phosphorylated by eye-PKC in vitro and, importantly, in the fly eye, as shown by mass spectrometry. Furthermore, transgenic expression of modified TRP bearing an Ala substitution leads to slow deactivation of the visual response similar to that of InaD(p215). These results suggest that the INAD macromolecular complex plays an essential role in termination of the light response by promoting efficient phosphorylation at Ser982 of TRP for fast deactivation of the visual signaling.


Assuntos
Proteínas de Drosophila/fisiologia , Drosophila/fisiologia , Proteínas do Olho/fisiologia , Olho/enzimologia , Proteína Quinase C/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Visão Ocular/fisiologia , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Drosophila/metabolismo , Técnicas In Vitro , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Fosforilação , Canais de Potencial de Receptor Transitório/química , Canais de Potencial de Receptor Transitório/genética
11.
J Biol Chem ; 278(24): 21576-83, 2003 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-12682077

RESUMO

Multiple PDZ domain protein 1 (MUPP1), a putative scaffolding protein containing 13 PSD-95, Dlg, ZO-1 (PDZ) domains, was identified by a yeast two-hybrid screen as a serotonin2C receptor (5-HT2C R)-interacting protein (Ullmer, C., Schmuck, K., Figge, A., and Lubbert, H. (1998) FEBS Lett. 424, 63-68). MUPP1 PDZ domain 10 (PDZ 10) associates with Ser458-Ser-Val at the carboxyl-terminal tail of the 5-HT2C R. Both Ser458 and Ser459 are phosphorylated upon serotonin stimulation of the receptor (Backstrom, J. R., Price, R. D., Reasoner, D. T., and Sanders-Bush, E. (2000) J. Biol. Chem. 275, 23620-23626). To investigate whether phosphorylation of these serines in the receptor regulates MUPP1 interaction, we used several approaches. First, we substituted the serines in the receptor carboxyl tail with aspartates to mimic phosphorylation (S458D, S459D, or S458D/S459D). Pull-down assays demonstrated that Asp mutations at Ser458 significantly decreased receptor tail interaction with PDZ 10. Next, serotonin treatment of 5-HT2C R/3T3 cells resulted in a dose-dependent reduction of receptor interaction with PDZ 10. Effects of serotonin on receptor-PDZ 10 binding could be blocked by pretreatment with a receptor antagonist. Alkaline phosphatase treatment reverses the effect of serotonin, indicating that agonist-induced phosphorylation at Ser458 resulted in a loss of MUPP1 association and also revealed a significant amount of basal phosphorylation of the receptor. We conclude that 5-HT2C R interaction with MUPP1 is dynamically regulated by phosphorylation at Ser458.


Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Receptores de Serotonina/metabolismo , Células 3T3 , Fosfatase Alcalina/farmacologia , Animais , Western Blotting , DNA/metabolismo , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Glutationa Transferase/metabolismo , Glicosilação , Proteínas de Membrana , Camundongos , Mutação , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Ligação Proteica , Biossíntese de Proteínas , Dobramento de Proteína , Estrutura Terciária de Proteína , Receptor 5-HT2C de Serotonina , Proteínas Recombinantes de Fusão/metabolismo , Serina/química , Serotonina/química , Valina/química
12.
J Biochem ; 132(4): 523-7, 2002 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12359065

RESUMO

Six protein kinase C (PKC) genes are present in Drosophila, comprising two classical PKCs (PKC53E and eye-PKC), two novel PKCs (PKC98E and PKCdelta), an atypical PKC (DaPKC), and a PKC-related kinase. Loss of function alleles affecting DaPKC and eye-PKC are available and their mutant phenotypes have been characterized. DaPKC is essential for early embryonic development because it regulates cell polarity and asymmetric cell division. Eye-PKC plays a role in the regulation of visual signaling, a G-protein coupled phospholipase Cbeta-mediated cascade. Both eye-PKC and DaPKC are differentially localized through tethering to multimolecular complexes. DaPKC interacts with partitioning-defective 3 (Par-3) and Par-6 proteins, which contain PDZ (PSD95, DLG, ZO-1) domains. Similarly, eye-PKC is anchored to a PDZ domain containing scaffolding protein INAD. Characterization of these two PKCs in Drosophila revealed a universal mechanism by which PKC is tethered to specific protein complexes for participation in distinct signal transduction processes.


Assuntos
Proteínas de Drosophila/fisiologia , Drosophila/enzimologia , Proteínas de Insetos/fisiologia , Proteína Quinase C/fisiologia , Animais , Polaridade Celular/fisiologia , Bases de Dados Genéticas , Drosophila/genética , Drosophila/fisiologia , Proteínas de Drosophila/química , Proteínas do Olho/química , Proteínas do Olho/fisiologia , Humanos , Proteínas de Insetos/química , Isoenzimas/química , Isoenzimas/fisiologia , Proteína Quinase C/química , Estrutura Terciária de Proteína/fisiologia , Transdução de Sinais , Visão Ocular/fisiologia
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