In recent years, thyrotropin-releasing hormone (TRH) and its analogs, including taltirelin (TAL), have demonstrated a range of effects on the central nervous system that represent potential therapeutic agents for the treatment of various neurological disorders, including neurodegenerative diseases. However, the molecular mechanisms of their actions remain poorly understood. In this study, we investigated phosphosignaling dynamics in pituitary GH1 cells affected by TRH and TAL and the putative role of β-arrestin2 in mediating these effects. Our results revealed widespread alterations in many phosphosignaling pathways involving signal transduction via small GTPases, MAP kinases, Ser/Thr- and Tyr-protein kinases, Wnt/β-catenin, and members of the Hippo pathway. The differential TRH- or TAL-induced phosphorylation of numerous proteins suggests that these ligands exhibit some degree of biased agonism at the TRH receptor. The different phosphorylation patterns induced by TRH or TAL in β-arrestin2-deficient cells suggest that the β-arrestin2 scaffold is a key factor determining phosphorylation events after TRH receptor activation. Our results suggest that compounds that modulate kinase and phosphatase activity can be considered as additional adjuvants to enhance the potential therapeutic value of TRH or TAL.
β-Arrestins are known to play a crucial role in GPCR-mediated transmembrane signaling processes. However, there are still many unanswered questions, especially those concerning the presumed similarities and differences of β-arrestin isoforms. Here, we examined the roles of β-arrestin 1 and β-arrestin 2 at different levels of μ-opioid receptor (MOR)-regulated signaling, including MOR mobility, internalization of MORs, and adenylyl cyclase (AC) activity. For this purpose, naïve HEK293 cells or HEK293 cells stably expressing YFP-tagged MOR were transfected with appropriate siRNAs to block in a specific way the expression of β-arrestin 1 or β-arrestin 2. We did not find any significant differences in the ability of β-arrestin isoforms to influence the lateral mobility of MORs in the plasma membrane. Using FRAP and line-scan FCS, we observed that knockdown of both β-arrestins similarly increased MOR lateral mobility and diminished the ability of DAMGO and endomorphin-2, respectively, to enhance and slow down receptor diffusion kinetics. However, β-arrestin 1 and β-arrestin 2 diversely affected the process of agonist-induced MOR endocytosis and exhibited distinct modulatory effects on AC function. Knockdown of β-arrestin 1, in contrast to β-arrestin 2, more effectively suppressed forskolin-stimulated AC activity and prevented the ability of activated-MORs to inhibit the enzyme activity. Moreover, we have demonstrated for the first time that β-arrestin 1, and partially β-arrestin 2, may somehow interact with AC and that this interaction is strongly supported by the enzyme activation. These data provide new insights into the functioning of β-arrestin isoforms and their distinct roles in GPCR-mediated signaling.
The interactions between TRPV1 and µ-opioid receptors (MOR) have recently attracted much attention because these two receptors play important roles in pain pathways and can apparently modulate each other's functioning. However, the knowledge about signaling interactions and crosstalk between these two receptors is still limited. In this study, we investigated the mutual interactions between MOR and TRPV1 shortly after their activation in HEK293 cells expressing these two receptors. After activation of one receptor we observed significant changes in the other receptor's lateral mobility and vice versa. However, the changes in receptor movement within the plasma membrane were not connected with activation of the other receptor. We also observed that plasma membrane β-arrestin 2 levels were altered after treatment with agonists of both these receptors. Knockdown of β-arrestin 2 blocked all changes in the lateral mobility of both receptors. Furthermore, we found that β-arrestin 2 can play an important role in modulating the effectiveness of ERK1/2 phosphorylation after activation of MOR in the presence of TRPV1. These data suggest that β-arrestin 2 and ERK1/2 are important mediators between these two receptors and their signaling pathways. Collectively, MOR and TRPV1 can mutually affect each other's behavior and β-arrestin 2 apparently plays a key role in the bidirectional crosstalk between these two receptors in the plasma membrane.
- MeSH
- arrestiny metabolismus MeSH
- beta arrestin 2 metabolismus fyziologie MeSH
- beta arrestiny metabolismus MeSH
- buněčná membrána metabolismus fyziologie MeSH
- fosforylace MeSH
- HEK293 buňky MeSH
- kationtové kanály TRPV metabolismus fyziologie MeSH
- lidé MeSH
- MAP kinasový signální systém fyziologie MeSH
- morfin metabolismus MeSH
- opioidní analgetika metabolismus MeSH
- receptory opiátové mu metabolismus fyziologie MeSH
- receptory opiátové metabolismus MeSH
- signální transdukce MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
OBJECTIVES: Extensive research has been dedicated to elucidating the mechanisms of signal transduction through different G protein-coupled receptors (GPCRs). However, relatively little is known about the regulation of receptor movement within the cell membrane upon ligand binding. In this study we focused our attention on the thyrotropin-releasing hormone (TRH) receptor that typically couples to Gq/11 proteins. METHODS: We monitored receptor diffusion in the plasma membrane of HEK293 cells stably expressing yellow fluorescent protein (YFP)-tagged TRH receptor (TRHR-YFP) by fluorescence recovery after photobleaching (FRAP). RESULTS: FRAP analysis indicated that the lateral movement of the TRH receptor was markedly reduced upon TRH binding as the value of its diffusion coefficient fell down by 55%. This effect was prevented by the addition of the TRH receptor antagonist midazolam. We also found that siRNA-mediated knockdown of Gq/11α, Gβ, β-arrestin2 and phospholipase Cβ1, but not of Giα1, β-arrestin1 or G protein-coupled receptor kinase 2, resulted in a significant decrease in the rate of TRHR-YFP diffusion, indicating the involvement of the former proteins in the regulation of TRH receptor behavior. The observed partial reduction of the TRHR-YFP mobile fraction caused by down-regulation of Giα1 and β-arrestin1 suggests that these proteins may also play distinct roles in THR receptor-mediated signaling. CONCLUSION: These results demonstrate for the first time that not only agonist binding but also abundance of some signaling proteins may strongly affect TRH receptor dynamics in the plasma membrane.
- MeSH
- beta arrestiny chemie genetika MeSH
- buněčná membrána účinky léků MeSH
- FRAP MeSH
- HEK293 buňky MeSH
- hormon uvolňující thyreotropin chemie metabolismus MeSH
- kinasa 2 receptorů spřažených s G-proteiny chemie MeSH
- lidé MeSH
- ligandy MeSH
- midazolam farmakologie MeSH
- proteiny vázající GTP - alfa-podjednotky Gi-Go chemie genetika MeSH
- receptory thyroliberinu agonisté antagonisté a inhibitory chemie genetika MeSH
- signální transdukce účinky léků genetika MeSH
- vazba proteinů účinky léků MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
β-Adrenergic signaling plays an important role in regulating diverse brain functions and alterations in this signaling have been observed in different neuropathological conditions. In this study, we investigated the effect of a 10-day treatment with high doses of morphine (10 mg/kg per day) on major components and functional state of the β-adrenergic receptor (β-AR) signaling system in the rat cerebral cortex. β-ARs were characterized by radioligand binding assays and amounts of various G protein subunits, adenylyl cyclase (AC) isoforms, G protein-coupled receptor kinases (GRKs), and β-arrestin were examined by Western blot analysis. AC activity was determined as a measure of functionality of the signaling system. We also assessed the partitioning of selected signaling proteins between the lipid raft and non-raft fractions prepared from cerebrocortical plasma membranes. Morphine treatment resulted in a significant upregulation of β-ARs, GRK3, and some AC isoforms (AC-I, -II, and -III). There was no change in quantity of G proteins and some other signaling molecules (AC-IV, AC-V/VI, GRK2, GRK5, GRK6, and β-arrestin) compared with controls. Interestingly, morphine exposure caused a partial redistribution of β-ARs, Gsα, Goα, and GRK2 between lipid rafts and bulk plasma membranes. Spatial localization of other signaling molecules within the plasma membrane was not changed. Basal as well as fluoride- and forskolin-stimulated AC activities were not significantly different in membrane preparations from control and morphine-treated animals. However, AC activity stimulated by the beta-AR agonist isoprenaline was markedly increased. This is the first study to demonstrate lipid raft association of key components of the cortical β-AR system and its sensitivity to morphine.
- MeSH
- adenylátcyklasy genetika metabolismus MeSH
- beta arrestiny genetika metabolismus MeSH
- beta-adrenergní receptory metabolismus MeSH
- kinasa 3 receptorů spřažených s G-proteiny genetika metabolismus MeSH
- krysa rodu rattus MeSH
- membránové mikrodomény metabolismus MeSH
- morfin farmakologie MeSH
- mozková kůra účinky léků metabolismus MeSH
- narkotika farmakologie MeSH
- potkani Wistar MeSH
- signální transdukce * MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
AT(1) receptor (AT1R) blockade prevents physiological cardiac hypertrophy induced by resistance training. Also, our group showed that a single bout of resistance exercise (RE) activates the AKT/mTOR which was also inhibited by AT1R blocker. Here, we investigated whether IGF1-receptor (IGF1-R) and MAPKs were also activated after a single bout of RE. Wistar rats were divided into Sedentary (Sed), Sedentary treated with losartan (Sed+LOS), Exercise (EX), and Exercise treated with losartan (EX+LOS). Cardiac tissue was obtained 5 and 30 min after 4 sets of 12 repetitions of squat exercise (80 % 1RM). We demonstrated that a single bout of RE did not induce IGF1-R tyrosine phosphorylation. ERK1/2 and P38 phosphorylation levels were elevated in the EX 5min and EX 30min groups however, only ERK1/2 was inhibited by losartan treatment (AT1R blocker). Next, we showed that beta-arrestin-2 expression increased 28 % in trained animals compared to sedentary group. Altogether, our results demonstrate that AT1R, but not IGF1-R, may exert the hypertrophic cardiac stimulus RE-induced. Also, activation of AKT/mTOR and ERK1/2 pathways may occur through the beta-arrestin-dependent pathway.
- MeSH
- beta arrestin 2 metabolismus MeSH
- blokátory receptorů AT1 pro angiotensin II farmakologie MeSH
- časové faktory MeSH
- extracelulárním signálem regulované MAP kinasy metabolismus MeSH
- interakce mezi receptory a ligandy MeSH
- kardiomegalie indukovaná tělesnou námahou * účinky léků MeSH
- kondiční příprava zvířat metody MeSH
- losartan farmakologie MeSH
- myokard metabolismus MeSH
- odporový trénink * MeSH
- potkani Wistar MeSH
- receptor angiotensinu typ 1 účinky léků metabolismus MeSH
- receptor IGF typ 1 metabolismus MeSH
- signální transdukce * účinky léků MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Nutrient availability controls the landscape of nutrient transporters present at the plasma membrane, notably by regulating their ubiquitylation and subsequent endocytosis. In yeast, this involves the Nedd4 ubiquitin ligase Rsp5 and arrestin-related trafficking adaptors (ARTs). ARTs are targeted by signaling pathways and warrant that cargo ubiquitylation and endocytosis appropriately respond to nutritional inputs. Here, we show that glucose deprivation regulates the ART protein Csr2/Art8 at multiple levels to trigger high-affinity glucose transporter endocytosis. Csr2 is transcriptionally induced in these conditions through the AMPK orthologue Snf1 and downstream transcriptional repressors. Upon synthesis, Csr2 becomes activated by ubiquitylation. In contrast, glucose replenishment induces CSR2 transcriptional shutdown and switches Csr2 to an inactive, deubiquitylated form. This glucose-induced deubiquitylation of Csr2 correlates with its phospho-dependent association with 14-3-3 proteins and involves protein kinase A. Thus, two glucose signaling pathways converge onto Csr2 to regulate hexose transporter endocytosis by glucose availability. These data illustrate novel mechanisms by which nutrients modulate ART activity and endocytosis.
- MeSH
- arrestin genetika metabolismus MeSH
- časové faktory MeSH
- endocytóza * MeSH
- genetická transkripce MeSH
- glukosa nedostatek MeSH
- jaderné proteiny genetika metabolismus MeSH
- mutace MeSH
- protein-serin-threoninkinasy metabolismus MeSH
- proteinfosfatasa 1 metabolismus MeSH
- proteinkinasy závislé na cyklickém AMP metabolismus MeSH
- proteiny 14-3-3 metabolismus MeSH
- proteiny přenášející monosacharidy genetika metabolismus MeSH
- regulace genové exprese u hub MeSH
- represorové proteiny metabolismus MeSH
- Saccharomyces cerevisiae - proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- ubikvitinace MeSH
- Publikační typ
- časopisecké články MeSH
- MeSH
- adenom hypofýzy vylučující růstový hormon genetika patologie terapie MeSH
- adenom genetika patologie terapie MeSH
- algoritmy MeSH
- antigen Ki-67 metabolismus MeSH
- arrestiny metabolismus MeSH
- beta arrestin 1 MeSH
- beta arrestiny MeSH
- cyklické peptidy terapeutické užití MeSH
- hormonální protinádorové látky terapeutické užití MeSH
- individualizovaná medicína MeSH
- intracelulární signální peptidy a proteiny metabolismus MeSH
- kadheriny metabolismus MeSH
- lidé MeSH
- mutace MeSH
- neurochirurgické výkony MeSH
- neúspěšná terapie MeSH
- oktreotid terapeutické užití MeSH
- prognóza MeSH
- raf kinasy metabolismus MeSH
- receptory somatostatinu metabolismus MeSH
- receptory spřažené s G-proteiny genetika MeSH
- somatostatin analogy a deriváty terapeutické užití MeSH
- tumor burden MeSH
- výsledek terapie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- souhrny MeSH
Many diseases of the nervous system are accompanied by alterations in synaptic functions. Synaptic plasticity mediated by the endogenous cannabinoid system involves the activation of the cannabinoid receptor 1 (CB1R). The principles of CB1R signaling must be understood in detail for its therapeutic exploration. We detected the Src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1 (SGIP1) as a novel CB1R partner. SGIP1 is functionally linked to clathrin-mediated endocytosis and its overexpression in animals leads to an energy regulation imbalance resulting in obesity. We report that SGIP1 prevents the endocytosis of activated CB1R and that it alters signaling via the CB1R in a biased manner. CB1R mediated G-protein activation is selectively influenced by SGIP1, β-arrestin associated signaling is changed profoundly, most likely as a consequence of the prevention of the receptor's internalization elicited by SGIP1.
- MeSH
- beta arrestin 2 metabolismus MeSH
- buněčná membrána účinky léků metabolismus MeSH
- endocytóza účinky léků fyziologie MeSH
- HEK293 buňky MeSH
- lidé MeSH
- MAP kinasový signální systém fyziologie MeSH
- mozek metabolismus MeSH
- myši MeSH
- neurony metabolismus MeSH
- potkani Wistar MeSH
- receptor kanabinoidní CB1 metabolismus MeSH
- Saccharomyces cerevisiae MeSH
- techniky dvojhybridového systému MeSH
- transfekce MeSH
- transportní proteiny genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
β-Arrestin is a scaffold protein that regulates signal transduction by seven transmembrane-spanning receptors. Among other functions it is also critically required for Wnt/β-catenin signal transduction. In the present study we provide for the first time a mechanistic basis for the β-arrestin function in Wnt/β-catenin signaling. We demonstrate that β-arrestin is required for efficient Wnt3a-induced Lrp6 phosphorylation, a key event in downstream signaling. β-Arrestin regulates Lrp6 phosphorylation via a novel interaction with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)-binding protein Amer1/WTX/Fam123b. Amer1 has been shown very recently to bridge Wnt-induced and Dishevelled-associated PtdIns(4,5)P2 production to the phosphorylation of Lrp6. Using fluorescence recovery after photobleaching we show here that β-arrestin is required for the Wnt3a-induced Amer1 membrane dynamics and downstream signaling. Finally, we show that β-arrestin interacts with PtdIns kinases PI4KIIα and PIP5KIβ. Importantly, cells lacking β-arrestin showed higher steady-state levels of the relevant PtdInsP and were unable to increase levels of these PtdInsP in response to Wnt3a. In summary, our data show that β-arrestins regulate Wnt3a-induced Lrp6 phosphorylation by the regulation of the membrane dynamics of Amer1. We propose that β-arrestins via their scaffolding function facilitate Amer1 interaction with PtdIns(4,5)P2, which is produced locally upon Wnt3a stimulation by β-arrestin- and Dishevelled-associated kinases.
- MeSH
- adaptorové proteiny signální transdukční genetika metabolismus MeSH
- arrestiny genetika metabolismus MeSH
- buněčná membrána metabolismus MeSH
- embryo savčí cytologie MeSH
- fibroblasty cytologie metabolismus MeSH
- fosfatidylinositol-4,5-difosfát metabolismus MeSH
- fosfoproteiny genetika metabolismus MeSH
- fosforylace MeSH
- fosfotransferasy s alkoholovou skupinou jako akceptorem genetika metabolismus MeSH
- HEK293 buňky MeSH
- konfokální mikroskopie MeSH
- kultivované buňky MeSH
- LDL receptor related protein 6 genetika metabolismus MeSH
- lidé MeSH
- myši knockoutované MeSH
- myši MeSH
- nádorové supresorové proteiny genetika metabolismus MeSH
- protein Wnt3A genetika metabolismus MeSH
- RNA interference MeSH
- vazba proteinů MeSH
- western blotting MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
