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Muscarinic acetylcholine receptors are prototypical G protein-coupled receptors (GPCRs), members of a large family of 7 transmembrane receptors mediating a wide variety of extracellular signals. We show here, in cultured cells and in a murine model, that the carboxyl terminal fragment of the muscarinic M2 receptor, comprising the transmembrane regions 6 and 7 (M2tail), is expressed by virtue of an internal ribosome entry site localized in the third intracellular loop. Single-cell imaging and import in isolated yeast mitochondria reveals that M2tail, whose expression is up-regulated in cells undergoing integrated stress response, does not follow the normal route to the plasma membrane, but is almost exclusively sorted to the mitochondria inner membrane: here, it controls oxygen consumption, cell proliferation, and the formation of reactive oxygen species (ROS) by reducing oxidative phosphorylation. Crispr/Cas9 editing of the key methionine where cap-independent translation begins in human-induced pluripotent stem cells (hiPSCs), reveals the physiological role of this process in influencing cell proliferation and oxygen consumption at the endogenous level. The expression of the C-terminal domain of a GPCR, capable of regulating mitochondrial function, constitutes a hitherto unknown mechanism notably unrelated to its canonical signaling function as a GPCR at the plasma membrane. This work thus highlights a potential novel mechanism that cells may use for controlling their metabolism under variable environmental conditions, notably as a negative regulator of cell respiration. The muscarinic M2 receptor is a prototypical G-protein-coupled receptor that mediates a wide range of extracellular signals. These authors show that the C-terminal fragment of M2 localizes to the mitochondrial inner membrane and is upregulated upon stress exposure, reducing cellular respiration and ROS to protect cells against variable environmental conditions. [ABSTRACT FROM AUTHOR]

Unstructured regions in functional proteins have gained attention in recent years due to advancements in informatics tools and biophysical methods. G protein-coupled receptors (GPCRs), a large family of cell surface receptors, contain unstructured regions in the form of the i3 loop and C-terminus. This review provides an overview of the functional significance of these regions in GPCRs. GPCRs transmit signals from the extracellular environment to the cell interior, regulating various physiological processes. The i3 loop, located between the fifth and sixth transmembrane helices, and the C-terminus, connected to the seventh transmembrane helix, are determinant of interactions with G proteins and with other intracellular partners such as arrestins. Recent studies demonstrate that the i3 loop and C-terminus play critical roles in allosterically regulating GPCR activation. They can act as autoregulators, adopting conformations that, by restricting G protein access, modulate receptor coupling specificity. The length and unstructured nature of the i3 loop and C-terminus provide unique advantages in GPCR interactions with intracellular protein partners. They act as "fishing lines", expanding the radius of interaction and enabling GPCRs to tether scaffolding proteins, thus facilitating receptor stability during cell membrane movements. Additionally, the i3 loop may be involved in domain swapping between GPCRs, generating novel receptor dimers with distinct binding and coupling characteristics. Overall, the i3 loop and C-terminus are now widely recognized as crucial elements in GPCR function and regulation. Understanding their functional roles enhances our comprehension of GPCR structure and signaling complexity and holds promise for advancements in receptor pharmacology and drug development. [ABSTRACT FROM AUTHOR]

The architecture of eukaryotic cells is defined by extensive membrane-delimited compartments, which entails separate metabolic processes that would otherwise interfere with each other, leading to functional differences between cells. G protein-coupled receptors (GPCRs) are the largest class of cell surface receptors, and their signal transduction is traditionally viewed as a chain of events initiated from the plasma membrane. Furthermore, their intracellular trafficking, internalization, and recycling were considered only to regulate receptor desensitization and cell surface expression. On the contrary, accumulating data strongly suggest that GPCRs also signal from intracellular compartments. GPCRs localize in the membranes of endosomes, nucleus, Golgi and endoplasmic reticulum apparatuses, mitochondria, and cell division compartments. Importantly, from these sites they have shown to orchestrate multiple signals that regulate different cell pathways. In this review, we summarize the current knowledge of this fascinating phenomenon, explaining how GPCRs reach the intracellular sites, are stimulated by the endogenous ligands, and their potential physiological/pathophysiological roles. Finally, we illustrate several mechanisms involved in the modulation of the compartmentalized GPCR signaling by drugs and endogenous ligands. Understanding how GPCR signaling compartmentalization is regulated will provide a unique opportunity to develop novel pharmaceutical approaches to target GPCRs and potentially lead the way towards new therapeutic approaches. [ABSTRACT FROM AUTHOR]

Herein we describe the in vitro and in vivo activity of FK228 (Romidepsin), an inhibitor of class I HDACs, in counteracting and radiosensitizing embryonal (ERMS, fusion-negative) and alveolar (ARMS, fusion-positive) rhabdomyosarcoma (RMS). RH30 (ARMS, fusion-positive) and RD (ERMS, fusion-negative) cell lines and human multipotent mesenchymal stromal cells (HMSC) were used. Flow cytometry analysis, RT-qPCR, western blotting and enzymatic assays were performed. Irradiation was delivered by using an x-6 MV photon linear accelerator. FK228 (1.2 mg/kg) in vivo activity, combined or not with radiation therapy (2 Gy), was assessed in murine xenografts. Compared to HMSC, RMS expressed low levels of class I HDACs. In vitro, FK228, as single agents, reversibly downregulated class I HDACs expression and activity and induced oxidative stress, DNA damage and a concomitant growth arrest associated with PARP-1-mediated transient non-apoptotic cell death. Surviving cells upregulated the expression of cyclin A, B, D1, p27, Myc and activated PI3K/Akt/mTOR and MAPK signaling, known to be differently involved in cancer chemoresistance. Interestingly, while no radiosensitizing effects were detected, in vitro or in vivo, on RD cells, FK228 markedly radiosensitized RH30 cells by impairing antioxidant and DSBs repair pathways in vitro. Further, FK228 when combined with RT in vivo significantly reduced tumor mass in mouse RH30 xenografts. FK228 did not show antitumor activity as a single agent whilst its combination with RT resulted in radiosensitization of fusion-positive RMS cells, thus representing a possible strategy for the treatment of the most aggressive RMS subtype. [ABSTRACT FROM AUTHOR]

Purpose: Radiation therapy (RT), by using ionizing radiation (IR), destroys cancer cells inducing DNA damage. Despite several studies are continuously performed to identify the best curative dose of IR, the role of dose-rate, IR delivered per unit of time, on tumor control is still largely unknown. Materials and methods: Rhabdomyosarcoma (RMS) and prostate cancer (PCa) cell lines were irradiated with 2 or 10 Gy delivered at dose-rates of 1.5, 2.5, 5.5 and 10.1 Gy/min. Cell-survival rate and cell cycle distribution were evaluated by clonogenic assays and flow cytometry, respectively. The production of reactive oxygen species (ROS) was detected by cytometry. Quantitative polymerase chain reaction assessed the expression of anti-oxidant-related factors including NRF2, SODs, CAT and GPx4 and miRNAs (miR-22, -126, -210, -375, -146a, -34a). Annexin V and caspase-8, -9 and -3 activity were assessed to characterize cell death. Senescence was determined by assessing β-galactosidase (SA-β-gal) activity. Immunoblotting was performed to assess the expression/activation of: i) phosphorylated H2AX (γ-H2AX), markers of DNA double strand breaks (DSBs); ii) p19Kip1/Cip1, p21Waf1/Cip1 and p27Kip1/Cip1, senescence-related-markers; iii) p62, LC3-I and LC3-II, regulators of autophagy; iv) ATM, RAD51, DNA-PKcs, Ku70 and Ku80, mediators of DSBs repair. Results: Low dose-rate (LDR) more efficiently induced apoptosis and senescence in RMS while high dose-rate (HDR) necrosis in PCa. This paralleled with a lower ability of LDR-RMS and HDR-PCa irradiated cells to activate DSBs repair. Modulating the dose rate did not differently affect the anti-oxidant ability of cancer cells. Conclusion: The present results indicate that a stronger cytotoxic effect was induced by modulating the dose-rate in a cancer cell-dependent manner, this suggesting that choose the dose-rate based on the individual patient's tumor characteristics could be strategic for effective RT exposures. [ABSTRACT FROM AUTHOR]

Erythropoietin-producing hepatocellular receptors (Eph) promote the onset and sustain the progression of cancers such as colorectal cancer (CRC), in which the A2 subtype of Eph receptor expression has been shown to correlate with a poor prognosis and has been identified as a promising therapeutic target. Herein, we investigated, in vitro and in vivo, the effects of treatment with GLPG1790, a potent pan-Eph inhibitor. The small molecule has selective activity against the EphA2 isoform in human HCT116 and HCT15 CRC cell lines expressing a constitutively active form of RAS concurrently with a wild-type or mutant form of p53, respectively. GLPG1790 reduced EPHA2 phosphorylation/activation and induced G1/S cell-cycle growth arrest by downregulating the expression of cyclin E and PCNA, while upregulating p21Waf1/Cip1 and p27Cip/Kip. The inhibition of ephrin signaling induced quiescence in HCT15 and senescence in HCT116 cells. While investigating the role of CRC-related, pro-oncogenic p53 and RAS pathways, we found that GLPG1790 upregulated p53 expression and that silencing p53 or inhibiting RAS (human rat sarcoma)/ERKs (extracellular signal-regulated kinase) signaling restrained the ability of GLPG1790 to induce senescence in HCT116 cells. On the other hand, HCT15 silencing of p53 predisposed cells to GLPG1790-induced senescence, whilst no effects of ERK inhibition were observed. Finally, GLPG1790 hindered the epithelial-mesenchymal transition, reduced the migratory capacities of CRC, and affected tumor formation in xenograft models in vivo more efficiently using HCT116 than HCT15 for xenografts. Taken together, our data suggest the therapeutic potential of GLPG1790 as a signal transduction-based therapeutic strategy in to treat CRC. [ABSTRACT FROM AUTHOR]

Truncated or shorter forms of G protein-coupled receptors (GPCRs), originating by alternative splicing, have been considered physiologically irrelevant for a rather long time. Nevertheless, it is now recognized that alternative splicing variants of GPCRs greatly increase the total number of receptor isoforms and can regulate receptor trafficking and signalling. Furthermore, dimerization of these truncated variants with other receptors concurs to expand receptor diversity. Highly truncated variants of GPCRs, typically, are retained in the endoplasmic reticulum (ER) and by heteromerization prevent the wild-type receptor to reach the plasma membrane, exerting a dominant-negative effect on its function. This can be responsible for some pathological conditions but in some other cases, it can offer protection from a disease because the expression of the receptor, that is necessary for binding an infectious agent, is attenuated. Here, we propose a possible new mechanism of creation of truncated GPCR variants through an internal ribosome entry site (IRES), a nucleotide sequence that allows cap independent translation of proteins by recruiting the ribosome in proximity of an internal initiation codon. We suggest that an IRES, situated in the third cytoplasmic loop, could be responsible for the translation of the last two transmembrane (TM) regions of the muscarinic M2 receptor. IRES driven expression of this C-terminal part of the muscarinic M2 receptor could represent a novel and additional mechanism of receptor regulation. [ABSTRACT FROM AUTHOR]

Recent data indicates that prolonged bright light exposure of rats induces production of neuromelanin and reduction of tyrosine hydroxylase positive neurons in the substantia nigra. This effect was the result of direct light reaching the substantia nigra and not due to alteration of circadian rhythms. Here, we measured the spectrum of light reaching the substantia nigra in rats and analysed the pathway that light may take to reach this deep brain structure in humans. Wavelength range and light intensity, emitted from a fluorescent tube, were measured, using a stereotaxically implanted optical fibre in the rat mesencephalon. The hypothetical path of environmental light from the eye to the substantia nigra in humans was investigated by computed tomography and magnetic resonance imaging. Light with wavelengths greater than 600 nm reached the rat substantia nigra, with a peak at 709 nm. Eyes appear to be the gateway for light to the mesencephalon since covering the eyes with aluminum foil reduced light intensity by half. Using computed tomography and magnetic resonance imaging of a human head, we identified the eye and the superior orbital fissure as possible gateways for environmental light to reach the mesencephalon. [ABSTRACT FROM AUTHOR]

Information flow from a source to a receiver becomes informative when the recipient can process the signal into a meaningful form. Information exchange and interpretation is essential in biology and understanding how cells integrate signals from a variety of information-coding molecules into complex orchestrated responses is a major challenge for modern cell biology. In complex organisms, cell to cell communication occurs mostly through neurotransmitters and hormones, and receptors are responsible for signal recognition at the membrane level and information transduction inside the cell. The G protein-coupled receptors (GPCRs) are the largest family of membrane receptors, with nearly 800 genes coding for these proteins. The recognition that GPCRs may physically interact with each other has led to the hypothesis that their dimeric state can provide the framework for temporal coincidence in signaling pathways. Furthermore, the formation of GPCRs higher order oligomers provides the structural basis for organizing distinct cell compartments along the plasma membrane where confined increases in second messengers may be perceived and discriminated. Here, we summarize evidence that supports these conjectures, fostering new ideas about the physiological role played by receptor homo- and hetero-oligomerization in cell biology. [ABSTRACT FROM AUTHOR]

Atypical antipsychotics (AAPs) are commonly prescribed medications to treat schizophrenia, bipolar disorders and other psychotic disorders. However, they might cause metabolic syndrome (MetS) in terms of weight gain, dyslipidemia, type 2 diabetes (T2D), and high blood pressure, which are responsible for reduced life expectancy and poor adherence. Importantly, there is clear evidence that early metabolic disturbances can precede weight gain, even if the latter still remains the hallmark of AAPs use. In fact, AAPs interfere profoundly with glucose and lipid homeostasis acting mostly on hypothalamus, liver, pancreatic β-cells, adipose tissue, and skeletal muscle. Their actions on hypothalamic centers via dopamine, serotonin, acetylcholine, and histamine receptors affect neuropeptides and 5′AMP-activated protein kinase (AMPK) activity, thus producing a supraphysiological sympathetic outflow augmenting levels of glucagon and hepatic glucose production. In addition, altered insulin secretion, dyslipidemia, fat deposition in the liver and adipose tissues, and insulin resistance become aggravating factors for MetS. In clinical practice, among AAPs, olanzapine and clozapine are associated with the highest risk of MetS, whereas quetiapine, risperidone, asenapine and amisulpride cause moderate alterations. The new AAPs such as ziprasidone, lurasidone and the partial agonist aripiprazole seem more tolerable on the metabolic profile. However, these aspects must be considered together with the differences among AAPs in terms of their efficacy, where clozapine still remains the most effective. Intriguingly, there seems to be a correlation between AAP's higher clinical efficacy and increase risk of metabolic alterations. Finally, a multidisciplinary approach combining psychoeducation and therapeutic drug monitoring (TDM) is proposed as a first-line strategy to avoid the MetS. In addition, pharmacological treatments are discussed as well. [ABSTRACT FROM AUTHOR]

Schizophrenia was first described by Emil Krapelin in the 19th century as one of the major mental illnesses causing disability worldwide. Since the introduction of chlorpromazine in 1952, strategies aimed at modifying the activity of dopamine receptors have played a major role for the treatment of schizophrenia. The introduction of atypical antipsychotics with clozapine broadened the range of potential targets for the treatment of this psychiatric disease, as they also modify the activity of the serotoninergic receptors. Interestingly, all marketed drugs for schizophrenia bind to the orthosteric binding pocket of the receptor as competitive antagonists or partial agonists. In recent years, a strong effort to develop allosteric modulators as potential therapeutic agents for schizophrenia was made, mainly for the several advantages in their use. In particular, the allosteric binding sites are topographically distinct from the orthosteric pockets, and thus drugs targeting these sites have a higher degree of receptor subunit specificity. Moreover, "pure" allosteric modulators maintain the temporal and spatial fidelity of native orthosteric ligand. Furthermore, allosteric modulators have a "ceiling effect", and their modulatory effect is saturated above certain concentrations. In this review, we summarize the progresses made in the identification of allosteric drugs for dopamine and serotonin receptors, which could lead to a new generation of atypical antipsychotics with a better profile, especially in terms of reduced side effects. [ABSTRACT FROM AUTHOR]