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.
- MeSH
- Adenylyl Cyclases * metabolism MeSH
- beta-Arrestin 1 metabolism MeSH
- beta-Arrestin 2 metabolism MeSH
- beta-Arrestins metabolism MeSH
- HEK293 Cells MeSH
- Humans MeSH
- Receptors, Opioid, mu * metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
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
- Arrestins metabolism MeSH
- beta-Arrestin 2 metabolism physiology MeSH
- beta-Arrestins metabolism MeSH
- Cell Membrane metabolism physiology MeSH
- Phosphorylation MeSH
- HEK293 Cells MeSH
- TRPV Cation Channels metabolism physiology MeSH
- Humans MeSH
- MAP Kinase Signaling System physiology MeSH
- Morphine metabolism MeSH
- Analgesics, Opioid metabolism MeSH
- Receptors, Opioid, mu metabolism physiology MeSH
- Receptors, Opioid metabolism MeSH
- Signal Transduction MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article 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-Arrestins chemistry genetics MeSH
- Cell Membrane drug effects MeSH
- Fluorescence Recovery After Photobleaching MeSH
- HEK293 Cells MeSH
- Thyrotropin-Releasing Hormone chemistry metabolism MeSH
- G-Protein-Coupled Receptor Kinase 2 chemistry MeSH
- Humans MeSH
- Ligands MeSH
- Midazolam pharmacology MeSH
- GTP-Binding Protein alpha Subunits, Gi-Go chemistry genetics MeSH
- Receptors, Thyrotropin-Releasing Hormone agonists antagonists & inhibitors chemistry genetics MeSH
- Signal Transduction drug effects genetics MeSH
- Protein Binding drug effects MeSH
- Binding Sites MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article 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
- Adenylyl Cyclases genetics metabolism MeSH
- beta-Arrestins genetics metabolism MeSH
- Receptors, Adrenergic, beta metabolism MeSH
- G-Protein-Coupled Receptor Kinase 3 genetics metabolism MeSH
- Rats MeSH
- Membrane Microdomains metabolism MeSH
- Morphine pharmacology MeSH
- Cerebral Cortex drug effects metabolism MeSH
- Narcotics pharmacology MeSH
- Rats, Wistar MeSH
- Signal Transduction * MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article 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 metabolism MeSH
- Angiotensin II Type 1 Receptor Blockers pharmacology MeSH
- Time Factors MeSH
- Extracellular Signal-Regulated MAP Kinases metabolism MeSH
- Receptor Cross-Talk MeSH
- Cardiomegaly, Exercise-Induced * drug effects MeSH
- Physical Conditioning, Animal methods MeSH
- Losartan pharmacology MeSH
- Myocardium metabolism MeSH
- Resistance Training * MeSH
- Rats, Wistar MeSH
- Receptor, Angiotensin, Type 1 drug effects metabolism MeSH
- Receptor, IGF Type 1 metabolism MeSH
- Signal Transduction * drug effects MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- MeSH
- Growth Hormone-Secreting Pituitary Adenoma genetics pathology therapy MeSH
- Adenoma genetics pathology therapy MeSH
- Algorithms MeSH
- Ki-67 Antigen metabolism MeSH
- Arrestins metabolism MeSH
- beta-Arrestin 1 MeSH
- beta-Arrestins MeSH
- Peptides, Cyclic therapeutic use MeSH
- Antineoplastic Agents, Hormonal therapeutic use MeSH
- Precision Medicine MeSH
- Intracellular Signaling Peptides and Proteins metabolism MeSH
- Cadherins metabolism MeSH
- Humans MeSH
- Mutation MeSH
- Neurosurgical Procedures MeSH
- Treatment Failure MeSH
- Octreotide therapeutic use MeSH
- Prognosis MeSH
- raf Kinases metabolism MeSH
- Receptors, Somatostatin metabolism MeSH
- Receptors, G-Protein-Coupled genetics MeSH
- Somatostatin analogs & derivatives therapeutic use MeSH
- Tumor Burden MeSH
- Treatment Outcome MeSH
- Check Tag
- Humans MeSH
- Publication type
- Overall 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 metabolism MeSH
- Cell Membrane drug effects metabolism MeSH
- Endocytosis drug effects physiology MeSH
- HEK293 Cells MeSH
- Humans MeSH
- MAP Kinase Signaling System physiology MeSH
- Brain metabolism MeSH
- Mice MeSH
- Neurons metabolism MeSH
- Rats, Wistar MeSH
- Receptor, Cannabinoid, CB1 metabolism MeSH
- Saccharomyces cerevisiae MeSH
- Two-Hybrid System Techniques MeSH
- Transfection MeSH
- Carrier Proteins genetics metabolism MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
