Constitutive Activation Research Articles

Improved Malignancy of Colon Cancer Cells at Gene Expression Level with Constitutive Activation of the Eukaryotic Elongation Factor 2 (eEF2) Under Nutrition Deficient Conditions.

Regulation of protein production in response to physiological signals is achieved through precise control of Eukaryotic Elongation Factor 2 (eEF2), whose distinct translocase function is crucial for cell survival. Phosphorylation of eEF2 at its Thr56 (T56) residue inactivates this function in translation. Using genetically modified paralogue of a colon cancer cell line, HCT116 which carries a point mutation at Ser595-to-Alanine in the eEF2 gene we were able to create a constitutively active form of eEF2. Then, we compared these cells with their wild-type counterparts under complete growth and nutrient deprivation conditions. The mutant forms were created by using CRISPR/Cas9-mediated gene cassette knock-in (KI) strategy. The samples were analyzed by a microarray based approach. Carrying out a WikiPath analysis, we identified distinct signaling pathways and gene expression patterns that are differentially regulated in the mutant cells (HCT116KI), but not in wild-type HCT116 (HCT116WT) cells, particularly under nutrient deprived conditions. Our results suggest that the constitutive activation of eEF2 enables increase in the malignant gene expression in colon cancer cells compared to the wild type counterparts under nutrient deprived conditions.

Fedratinib combined with ropeginterferon alfa-2b in patients with myelofibrosis (FEDORA): study protocol for a multicentre, open-label, Bayesian phase II trial

BackgroundMyelofibrosis (MF) is a clonal haematopoietic disease, with median overall survival for patients with primary MF only 6.5 years. The most frequent gene mutation found in patients is JAK2V617F, causing constitutive activation of the kinase and activation of downstream signalling. Fedratinib is an oral selective JAK2 inhibitor. It has shown activity in MF and is well-tolerated, but combination with other therapies is likely needed to achieve clonal remission. Combining a JAK2 inhibitor with an interferon may be synergistic, as haematopoietic cells are activated from quiescence (a typical kinase resistance mechanism) rendering them more sensitive to inhibition.Ropeginterferon alfa-2b is a next generation pegylated interferon-α-2b with high tolerability and clinical activity in patients with MF, however, evidence of tolerability and activity in combination with fedratinib is lacking in this setting. The aim of the FEDORA trial is to assess tolerability, safety, and activity of fedratinib with ropeginterferon alfa-2b in patients with MF who require treatment to justify further investigation in a phase III trial.MethodsFEDORA is a single arm, multicentre, open-label, Bayesian phase II trial to assess tolerability, safety, and activity of fedratinib with ropeginterferon alfa-2b aiming to recruit 30 patients. Patients with JAK2V617F positive primary or secondary MF, who are aged ≥ 18 years, have intermediate-1 with palpable splenomegaly of > 5cm, intermediate-2, or high-risk disease according to the Dynamic International Prognostic Scoring System (DIPSS), and who require treatment are eligible. The primary outcome is tolerability, whereby the combination is deemed intolerable in a patient if drug-related toxicities in the first four months of treatment lead to: either drug being discontinued; delays in treatment exceeding 28 consecutive days; or death. FEDORA uses a within-patient dose escalation regimen to ensure each patient reaches a personalised dose combination that is acceptable.DiscussionFEDORA is using a Bayesian trial design and aims to provide evidence of the tolerability, safety, and activity of combining fedratinib with ropeginterferon alfa-2b upon which the decision as to whether a phase III trial is warranted will be based.Trial registrationEudraCT number: 2021–004056-42.ISRCTN: 88,102,629.

Loss-of-function GHSR variants are associated with short stature and low IGF-I.

The growth hormone (GH) secretagogue receptor, encoded by GHSR, is expressed on somatotrophs of the pituitary gland. Stimulation with its ligand ghrelin, as well as its constitutive activity, enhances GH secretion. Studies in knock-out mice suggest that heterozygous loss-of-function of GHSR is associated with decreased GH response to fasting, but patient observations in small case reports have been equivocal. To establish the phenotype of GHSR haploinsufficiency and their growth response to GH treatment. This case series includes 26 patients with short stature and heterozygous GHSR variants. Pathogenicity was studied in vitro using total protein levels, cell surface expression, and receptor activity in basal, stimulated and inhibited states. Ten different variants were identified, of which six were novel. Variants showed either partial or complete loss-of-function, primarily through loss of constitutive activity. Patients (4.0-15.1 years) had proportionate short stature (height -2.8±0.5 SDS), failure to thrive with low appetite (n=4), a mean serum insulin growth factor I (IGF-I) of -1.6±0.7 SDS, and a normal stimulated GH response. Nine patients received GH treatment, showing a height gain of 0.9±0.4 SDS after 1 year and 1.5±0.4 SDS after 2 years (n=5). This study combines phenotypical and functional data in a uniquely large group of children with short stature carrying GHSR variants, and shows their good response to GH treatment. The results strengthen the hypothesis of GHSR's role in GH secretion.

Development of high-performance inducible and secretory expression vector and host system for enhanced recombinant protein production

The production of lipopolysaccharide (LPS)-free recombinant proteins from culture supernatants is of great interest to biomedical research and industry. Due to the LPS-free cell wall structure and the well-defined secretion factor B (SecB)-dependent secretion pathway, Gram-positive bacteria are a superior alternative to Escherichia coli expression systems. However, the lack of inducible expression systems for high yields has been a bottleneck. To address this, we developed the pKS81 plasmid, featuring the uhpT (glucose-6-phosphate [G6P] transporter) promoter for high expression of recombinant proteins induced by extracellular G6P via a three-component hexose phosphate transport regulatory system (HptARS), the N-terminal SecB-dependent signal peptide sequence for recombinant protein secretion, and the C-terminal 8 × histidine tag for purification by nickel affinity chromatography. We also generated an expression host strain, Staphylococcus aureus LAC9, lacks the uhpT gene and harmful superantigen and leukotoxin genes, allowing for constitutive HptARS activation by extracellular G6P and increased safety, respectively. Using the pKS81 plasmid, we successfully achieved high yields of prokaryotic (staphylococcal leukotoxin E) and eukaryotic (human annexin A2 protein tagged with mouse IgG1) recombinant proteins, up to 900 mg/L. Our newly established inducible and secretory expression system provides for efficient production and easy purification of LPS-free recombinant proteins, making it valuable for biomedical research and industrial applications.

G Protein-Coupled Receptor 17 Inhibits Glucagon-like Peptide-1 Secretion via a Gi/o-Dependent Mechanism in Enteroendocrine Cells

Gut peptides, including glucagon-like peptide-1 (GLP-1), regulate metabolic homeostasis and have emerged as the basis for multiple state-of-the-art diabetes and obesity therapies. We previously showed that G protein-coupled receptor 17 (GPR17) is expressed in intestinal enteroendocrine cells (EECs) and modulates nutrient-induced GLP-1 secretion. However, the GPR17-mediated molecular signaling pathways in EECs have yet to be fully deciphered. Here, we expressed the human GPR17 long isoform (hGPR17L) in GLUTag cells, a murine EEC line, and we used the GPR17 synthetic agonist MDL29,951 together with pharmacological probes and genetic approaches to quantitatively assess the contribution of GPR17 signaling to GLP-1 secretion. Constitutive hGPR17L activity inhibited GLP-1 secretion, and MDL29,951 treatment further inhibited this secretion, which was attenuated by treatment with the GPR17 antagonist HAMI3379. MDL29,951 promoted both Gi/o and Gq protein coupling to mediate cyclic AMP (cAMP) and calcium signaling. hGPR17L regulation of GLP-1 secretion appeared to be Gq-independent and dependent upon Gi/o signaling, but was not correlated with MDL29,951-induced whole-cell cAMP signaling. Our studies revealed key signaling mechanisms underlying the role of GPR17 in regulating GLP-1 secretion and suggest future opportunities for pharmacologically targeting GPR17 with inverse agonists to maximize GLP-1 secretion.

The expression of pro-prion, a transmembrane isoform of the prion protein, leads to the constitutive activation of the canonical Wnt/β-catenin pathway to sustain the stem-like phenotype of human glioblastoma cells

The expression of pro-prion, a transmembrane isoform of the prion protein, leads to the constitutive activation of the canonical Wnt/β-catenin pathway to sustain the stem-like phenotype of human glioblastoma cells

Oncogenic PIK3CA corrupts growth factor signaling specificity.

Technical limitations have prevented understanding of how growth factor signals are encoded in distinct activity patterns of the phosphoinositide 3-kinase (PI3K)/AKT pathway, and how this is altered by oncogenic pathway mutations. We introduce a kinetic, single-cell framework for precise calculations of PI3K-specific information transfer for different growth factors. This features live-cell imaging of PI3K/AKT activity reporters and multiplexed CyTOF measurements of PI3K/AKT and RAS/ERK signaling markers over time. Using this framework, we found that the PIK3CAH1047R oncogene was not a simple, constitutive activator of the pathway as often presented. Dose-dependent expression of PIK3CAH1047R in human cervical cancer and induced pluripotent stem cells corrupted the fidelity of growth factor-induced information transfer, with preferential amplification of epidermal growth factor receptor (EGFR) signaling responses compared to insulin-like growth factor 1 (IGF1) and insulin receptor signaling. PIK3CAH1047R did not only shift these responses to a higher mean but also enhanced signaling heterogeneity. We conclude that oncogenic PIK3CAH1047R corrupts information transfer in a growth factor-dependent manner and suggest new opportunities for tuning of receptor-specific PI3K pathway outputs for therapeutic benefit.

YAP/TAZ mediates resistance to KRAS inhibitors through inhibiting proapoptosis and activating the SLC7A5/mTOR axis.

KRAS mutations are frequent in various human cancers. The development of selective inhibitors targeting KRAS mutations has opened a new era for targeted therapy. However, intrinsic and acquired resistance to these inhibitors remains a major challenge. Here, we found that cancer cells resistant to KRAS G12C inhibitors also display cross-resistance to other targeted therapies, such as inhibitors of RTKs or SHP2. Transcriptomic analyses revealed that the Hippo-YAP/TAZ pathway is activated in intrinsically resistant and acquired-resistance cells. Constitutive activation of YAP/TAZ conferred resistance to KRAS G12C inhibitors, while knockdown of YAP/TAZ or TEADs sensitized resistant cells to these inhibitors. This scenario was also observed in KRAS G12D-mutant cancer cells. Mechanistically, YAP/TAZ protects cells from KRAS inhibitor-induced apoptosis by downregulating the expression of proapoptotic genes such as BMF, BCL2L11, and PUMA, and YAP/TAZ reverses KRAS inhibitor-induced proliferation retardation by activating the SLC7A5/mTORC1 axis. We further demonstrated that dasatinib and MYF-03-176 notably enhance the efficacy of KRAS inhibitors by reducing SRC kinase activity and TEAD activity. Overall, targeting the Hippo-YAP/TAZ pathway has the potential to overcome resistance to KRAS inhibitors.

CaV3.2 T-type calcium channels contribute to CGRP- induced allodynia in a rodent model of experimental migraine

BackgroundMigraine is a painful neurological syndrome characterized by attacks of throbbing headache, of moderate to severe intensity, which is associated with photo- and phono- sensitivity as well as nausea and vomiting. It affects about 15% of the world’s population being 2–3 times more prevalent in females. The calcitonin gene-related peptide (CGRP) is a key mediator in the pathophysiology of migraine, and a significant advance in the field has been the development of anti-CGRP therapies. The trigeminal ganglion (TG) is thought to be an important site of action for these drugs. Moreover, experimental migraine can be induced by CGRP injection in the TG. The signaling pathway induced by CGRP in the TG is not fully understood, but studies suggest that voltage-gated calcium channels contribute to CGRP effects relevant to migraine.ObjectiveWe hypothesised that CGRP injection in the TG enhances CaV3.2 T-type calcium channel currents to contribute to the development of periorbital mechanical allodynia.ResultsA Co-Immunoprecipitation assay in tsA-201 cells revealed that CaV3.2 channels form a complex with RAMP-1, a component of the CGRP receptor. Constitutive CGRPR activity was able to inhibit CaV3.2 channels and induce a depolarizing shift in both activation and inactivation curves. Incubation of TG neurons with CGRP increased T-type current density by ~ 3.6 fold, an effect that was not observed in TG neurons from CaV3.2 knockout mice. Incubation of TG neurons with Z944, a pan T-type channel blocker, resulted in an approximately 80% inhibition of T-type currents. In vivo, this treatment abolished the development of periorbital mechanical allodynia induced by CGRP in male and female mice. Likewise, CaV3.2 knockout mice did not develop periorbital mechanical allodynia after intraganglionic CGRP injection. Finally, we demonstrated that the CGRP effect depends on the activation of its canonical GPCR, followed by protein kinase A activation.ConclusionThe present study suggests that CGRP modulates CaV3.2 in the TG, an effect possibly mediated by the canonical CGRP receptor and PKA activation. The increase in T-type currents in the TG may represent a contributing factor for the initiation and maintenance of the headache pain during migraine.Graphical abstract

DRD4 Interacts with TGF-β Receptors to Drive Colorectal Cancer Metastasis Independently of Dopamine Signaling Pathway.

The functional and pharmacological significance of dopamine receptor D4 (DRD4)in psychiatric and neurological disorders is well elucidated. However, the roles of DRD4 in colorectal cancer (CRC) remain unclear. This study observes a significant upregulation of DRD4 expression in clinical samples, which is negatively correlated with patient prognosis. In vitro, overexpression of DRD4 causes a constitutive activation of β-Arrestin2/PP2A/AKT independent of dopamine. Interestingly, this classical signaling pathway is not associated with the phenotype of DRD4-promoted migration and invasion in CRC cells. Instead, DRD4 interacts with transforming growth factor beta receptors (TGFBR1 and TGFBR2) to activate Smad2 phosphorylation and promote Smad2/Smad4 complex nucleus translocation. Then, SNAI1 and JAG1 are transcriptionally activated to induce epithelial-mesenchymal transition and enhance the metastatic potential of CRC. Notably, the COOH-terminal domain is identified as the key intracellular region for the pro-metastatic roles of DRD4. Furthermore, treatment with a TGFBR1 inhibitor combined with a BMP inhibitor effectively counteracts the pro-metastatic effects induced by DRD4 both in vitro and in vivo. In conclusion, these findings uncover an unconventional role for DRD4 beyond its classic function as a neurotransmitter receptor. The intracellular signaling of DRD4 interacting with TGFBR1 can be targeted pharmacologically for CRC therapy.

Inhibition of the PI3K-AKT-MTORC1 axis reduces the burden of the m.3243A>G mtDNA mutation by promoting mitophagy and improving mitochondrial function

ABSTRACT Mitochondrial DNA (mtDNA) encodes genes essential for oxidative phosphorylation. The m.3243A>G mutation causes severe disease, including myopathy, lactic acidosis and stroke-like episodes (MELAS) and is the most common pathogenic mtDNA mutation in humans. We have previously shown that the mutation is associated with constitutive activation of the PI3K-AKT-MTORC1 axis. Inhibition of this pathway in patient fibroblasts reduced the mutant load, rescued mitochondrial bioenergetic function and reduced glucose dependence. We have now investigated the mechanisms that select against the mutant mtDNA under these conditions. Basal macroautophagy/autophagy and lysosomal degradation of mitochondria were suppressed in the mutant cells. Pharmacological inhibition of any step of the PI3K-AKT-MTORC1 pathway activated mitophagy and progressively reduced m.3243A>G mutant load over weeks. Inhibition of autophagy with bafilomycin A1 or chloroquine prevented the reduction in mutant load, suggesting that mitophagy was necessary to remove the mutant mtDNA. Inhibition of the pathway was associated with metabolic remodeling – mitochondrial membrane potential and respiratory rate improved even before a measurable fall in mutant load and proved crucial for mitophagy. Thus, maladaptive activation of the PI3K-AKT-MTORC1 axis and impaired autophagy play a major role in shaping the presentation and progression of disease caused by the m.3243A>G mutation. Our findings highlight a potential therapeutic target for this otherwise intractable disease. Abbreviation: ΔΨm: mitochondrial membrane potential; 2DG: 2-deoxy-D-glucose; ANOVA: analysis of variance; ARMS-qPCR: amplification-refractory mutation system quantitative polymerase chain reaction; Baf A1: bafilomycin A1; BSA: bovine serum albumin; CQ: chloroquine; Cybrid: cytoplasmic hybrid; CYCS: cytochrome c, somatic; DCA: dichloroacetic acid; DMEM: Dulbecco’s modified Eagle’s medium; DMSO: dimethylsulfoxide; EGFP: enhanced green fluorescent protein; LC3B-I: carboxy terminus cleaved microtubule-associated protein 1 light chain 3 beta; LC3B-II: lipidated microtubule-associated protein 1 light chain 3 beta; LY: LY290042; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MELAS: mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes; MFC: mitochondrial fragmentation count; mt-Keima: mitochondrial-targeted mKeima; mtDNA: mitochondrial DNA/mitochondrial genome; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; OA: oligomycin+antimycin A; OxPhos: oxidative phosphorylation; DPBS: Dulbecco’s phosphate-buffered saline; PPARGC1A/PGC-1α: PPARG coactivator 1 alpha; PPARGC1B/PGC-1β: PPARG coactivator 1 beta; PI3K: phosphoinositide 3-kinase; PINK1: PTEN induced kinase 1; qPCR: quantitative polymerase chain reaction; RNA-seq: RNA sequencing; RP: rapamycin; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; WT: wild-type

Testicular sex cord-stromal tumors in mice with constitutive activation of PI3K and loss of Pten.

Testicular tumors are the most common malignancy of young men and tumors affecting the testis are caused by somatic mutations in germ or germ-like cells. The PI3K pathway is constitutively activated in about one third of testicular cancers. To investigate the role of the PI3K pathway in transforming stem-like cells in the testis, we investigated tumors derived from mice with post-natal, constitutive activation of PI3K signaling and homozygous deletion of tumor suppressor Pten, targeted to nestin expressing cells. Mice developed aggressive tumors, exhibiting heterogeneous histopathology and hemorrhaging. The tumors resemble the rare testis tumor type, testicular sex cord-stromal Leydig cell tumors. Single cell resolution spatial tissue analysis demonstrated that T-cells are the dominant tumor infiltrating immune cell type, with very few infiltrating macrophages observed in the tumor tissue, with CD8+ T-cells predominating. Further analysis showed that immune cells preferentially localize to or accumulate within stromal regions.

Essential strategies for the detection of constitutive and ligand-dependent Gi-directed activity of 7TM receptors using bioluminescence resonance energy transfer.

The constitutive (ligand-independent) signaling of G protein-coupled receptors (GPCRs) is being increasingly appreciated as an integral aspect of their function; however, it can be technically hard to detect for poorly characterized, e.g. orphan, receptors of the cAMP-inhibitory Gi-coupled (GiPCR) family. In this study, we delineate the optimal strategies for the detection of such activity across several GiPCRs in two cell lines. As our study examples, we chose two canonical GiPCRs - the constitutively active Smoothened and the ligand-activated CXCR4,-and one atypical GPCRs, the chemokine receptor ACKR3. We verified the applicability of three Bioluminescence Resonance Energy Transfer (BRET)-based assays - one measuring changes in intracellular cAMP, another in Gβγ/GRK3ct association and third in Gαi-Gβγ dissociation, - for assessing both constitutive and ligand-modulated activity of these receptors. We also revealed the possible caveats and sources of false positives, and proposed optimization strategies. All three types of assays confirmed the ligand-dependent activity of CXCR4, the controversial G protein incompetence of ACKR3, the constitutive Gi-directed activity of SMO, and its modulation by PTCH1. We also demonstrated that PTCH1 promotes SMO localization to the cell surface, thus enhancing its responsiveness not only to agonists but also to antagonists, which is a novel mechanism of regulation of a Class F GiPCR Smoothened.

Lymphoid enhancer-binding factor 1 (LEF1) immunostaining as a surrogate for β-catenin (CTNNB1) mutations

AimsMutations affecting exon 3 of the β-catenin (CTNNB1) gene result in constitutive activation of WNT signalling and are a diagnostic hallmark of several tumour entities including desmoid-type fibromatosis. They also...

Splicing junction-based classifier for the detection of abnormal constitutive activation of the KEAP1-NRF2 system

The KEAP1-NRF2 system plays a crucial role in responding to oxidative and electrophilic stress. Its dysregulation can cause the overexpression of downstream genes, a known cancer hallmark. Understanding and detecting abnormal KEAP1-NRF2 activity is essential for understanding disease mechanisms and identifying therapeutic targets. This study presents an approach that analyzes splicing patterns by a naive Bayes-based classifier to identify constitutive activation of the KEAP1-NRF2 system, focusing on the higher presence of abnormal splicing junctions as a subproduct of overexpression of downstream genes. Our splicing-based classifier demonstrated robust performance, reliably identifying activation of the KEAP1-NRF2 pathway across extensive datasets, including The Cancer Genome Atlas and the Sequence Read Archive. This shows the classifier’s potential to analyze hundreds of thousands of transcriptomes, highlighting its utility in broad-scale genomic studies and provides a new perspective on utilizing splicing aberrations caused by overexpression as diagnostic markers, offering potential improvements in diagnosis and treatment strategies.

Molecular profiling in MPN: who should have it and why?

Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) are a group of blood cancers that result from somatic mutations in hematopoietic stem cells, causing constitutive activation of JAK-STAT signaling pathways with consequent overproduction of 1 or more myeloid lineages. The initiating event in MPN pathogenesis is a genetic mutation, and consequently molecular profiling is central to the diagnosis, risk stratification, and, increasingly, monitoring of therapy response in persons with MPN. In this review we summarize current approaches to molecular profiling of classical MPNs (essential thrombocythemia, polycythemia vera, and myelofibrosis), using illustrative clinical case histories to demonstrate how genetic analysis is already fully integrated into MPN diagnostic classification and prognostic risk stratification. Molecular profiling can also be used in MPN to measure response to therapy both in clinical trials and increasingly in routine clinical practice. Taking a forward look, we discuss how molecular profiling in MPN might be used in the future to select specific molecularly targeted therapies and the role of additional genetic methodologies beyond mutation analysis.

Molecular insights into the activation mechanism of GPR156 in maintaining auditory function

The class C orphan G-protein-coupled receptor (GPCR) GPR156, which lacks the large extracellular region, plays a pivotal role in auditory function through Gi2/3. Here, we firstly demonstrate that GPR156 with high constitutive activity is essential for maintaining auditory function, and further reveal the structural basis of the sustained role of GPR156. We present the cryo-EM structures of human apo GPR156 and the GPR156–Gi3 complex, unveiling a small extracellular region formed by extracellular loop 2 (ECL2) and the N-terminus. The GPR156 dimer in both apo state and Gi3 protein-coupled state adopt a transmembrane (TM)5/6-TM5/6 interface, indicating the high constitutive activity of GPR156 in the apo state. Furthermore, C-terminus in G-bound subunit of GPR156 plays a dual role in promoting G protein binding within G-bound subunit while preventing the G-free subunit from binding to additional G protein. Together, these results explain how GPR156 constitutive activity is maintained through dimerization and provide a mechanistic insight into the sustained role of GPR156 in maintaining auditory function.

Pathogenesis and management of high molecular risk myeloproliferative neoplasms.

Classical myeloproliferative neoplasms (MPNs) are clonal stem cell disorders characterised by driver mutations that affect the constitutive activation of JAK-signalling. Additional mutations to an MPN-driver occur in a large number of patients and have been shown be associated with disease presentation and progression. In this review, we will outline the current hypotheses regarding how clonal evolution in MPN is thought to occur and the functional mechanisms as to how concomitant somatic mutations (i.e. mutations in genes other than the 'driver' genes) contribute to disease progression. We will discuss the definitions of high molecular risk MPN, provide an overview as to how concomitant mutations influence the clinical management of MPN and suggest how this rapidly developing genetic risk stratification can be utilised to improve clinical outcomes.

Partial activation of SIK3 delays the onset of wakefulness and alleviates hypersomnia due to the lack of protein kinase A-phosphorylation site.

Sleep/wakefulness is regulated by intracellular signaling pathways composed of protein kinases such as salt-inducible kinase 3 (Sik3). Sik3-deficiency in neurons decreases NREM sleep time and electroencephalogram (EEG) delta power during NREM sleep, while Sik3Slp mice lacking a protein kinase A (PKA)-phosphorylation site, S551, show hypersomnia phenotype. In this study, we examined how a phosphomimetic mutation of the 221st threonine residue (T221E), which provides a partial (weak) constitutive activity of the kinase, affects sleep/wakefulness and circadian behavior. We also examined the effect of T221E substitution on the hypersomnia phenotype of Sik3Slp mice. We examined the sleep/wake behavior of heterozygous and homozygous Sik3T221E mice and Sik3T221E;Slp mice using EEG and electromyogram (EMG) recording. We also examined the circadian behavior of Sik3T221E mice using a running wheel under the light-dark cycle and constant darkness. Heterozygous and homozygous Sik3T221E mice showed normal sleep time and sleep homeostatic responses. Homozygous Sik3T221E mice exhibited a delayed onset of wakefulness at the early dark phase and longer circadian periods. Sik3T221E;Slp mice showed decreased NREM sleep time and homeostatic responses compared to Sik3Slp mice. Our results suggest that the peak onset of wakefulness is sensitive to disturbed kinase activity of SIK3, and the relationship between phosphorylation at T221 and S551 is critical for regulating sleep need.

Structural insights into the high basal activity and inverse agonism of the orphan receptor GPR6 implicated in Parkinson's disease.

GPR6 is an orphan G protein-coupled receptor with high constitutive activity found in D2-type dopamine receptor-expressing medium spiny neurons of the striatopallidal pathway, which is aberrantly hyperactivated in Parkinson's disease. Here, we solved crystal structures of GPR6 without the addition of a ligand (a pseudo-apo state) and in complex with two inverse agonists, including CVN424, which improved motor symptoms in patients with Parkinson's disease in clinical trials. In addition, we obtained a cryo-electron microscopy structure of the signaling complex between GPR6 and its cognate Gs heterotrimer. The pseudo-apo structure revealed a strong density in the orthosteric pocket of GPR6 corresponding to a lipid-like endogenous ligand. A combination of site-directed mutagenesis, native mass spectrometry, and computer modeling suggested potential mechanisms for high constitutive activity and inverse agonism in GPR6 and identified a series of lipids and ions bound to the receptor. The structures and results obtained in this study could guide the rational design of drugs that modulate GPR6 signaling.