Structure-function relationships of the disease-linked A218T oxytocin receptor variant

AbstractVarious single nucleotide polymorphisms (SNPs) in the oxytocin receptor (OXTR) gene have been associated with behavioral traits, autism spectrum disorder (ASD) and other diseases. The non-synonymous SNP rs4686302 results in the OXTR variant A218T and has been linked to core characteristics of ASD, trait empathy and preterm birth. However, the molecular and intracellular mechanisms underlying those associations are still elusive. Here, we uncovered the molecular and intracellular consequences of this mutation that may affect the psychological or behavioral outcome of oxytocin (OXT)-treatment regimens in clinical studies, and provide a mechanistic explanation for an altered receptor function. We created two monoclonal HEK293 cell lines, stably expressing either the wild-type or A218T OXTR. We detected an increased OXTR protein stability, accompanied by a shift in Ca2+ dynamics and reduced MAPK pathway activation in the A218T cells. Combined whole-genome and RNA sequencing analyses in OXT-treated cells revealed 7823 differentially regulated genes in A218T compared to wild-type cells, including 429 genes being associated with ASD. Furthermore, computational modeling provided a molecular basis for the observed change in OXTR stability suggesting that the OXTR mutation affects downstream events by altering receptor activation and signaling, in agreement with our in vitro results. In summary, our study provides the cellular mechanism that links the OXTR rs4686302 SNP with genetic dysregulations associated with aspects of ASD.

Download Full-text

Excitatory Actions of GABA Increase BDNF Expression via a MAPK-CREB–Dependent Mechanism—A Positive Feedback Circuit in Developing Neurons

Journal of Neurophysiology ◽

10.1152/jn.2002.88.2.1005 ◽

2002 ◽

Vol 88 (2) ◽

pp. 1005-1015 ◽

Cited By ~ 102

Author(s):

Karl Obrietan ◽

Xiao-Bing Gao ◽

Anthony N. van den Pol

Keyword(s):

Transcription Factor ◽

Early Development ◽

Mapk Pathway ◽

Receptor Activation ◽

Mapk Cascade ◽

Bdnf Expression ◽

Creb Phosphorylation ◽

Pathway Activation ◽

Positive Feedback Circuit

During early neuronal development, GABA functions as an excitatory neurotransmitter, triggering membrane depolarization, action potentials, and the opening of plasma membrane Ca2+ channels. These excitatory actions of GABA lead to a number of changes in neuronal structure and function. Although the effects of GABA on membrane biophysics during early development have been well documented, little work has been done to examine the possible mechanisms underlying GABA-regulated plastic changes in the developing brain. This study focuses on GABA-regulated kinase activity and transcriptional control. We utilized a combination of Western blotting and immunocytochemical techniques to examine two potential downstream pathways regulated by GABA excitation: the p42/44 mitogen-activated protein kinase (MAPK) cascade and the transcription factor cyclic AMP response element binding protein (CREB). During early development of cultured hypothalamic neurons (5 days in vitro), stimulation with GABA triggered activation of the MAPK cascade and phosphorylation of CREB at Ser 133. These effects were mediated by the GABAAreceptor, since administration of the GABAAreceptor-specific agonist muscimol (50 μM) triggered pathway activation, and pretreatment with the GABAA-receptor specific antagonist bicuculline (20 μM) blocked pathway activation. Immunocytochemistry revealed a spatial and temporal correlation between activation of the MAPK cascade and CREB phosphorylation. Pretreatment with the MAPK/ERK kinase (MEK) inhibitor U0126 (10 μM) attenuated CREB phosphorylation, indicating that the MAPK pathway regulates that activation state of CREB. In contrast to the excitatory effects observed during early development, in more mature neurons, GABA functions as an inhibitory transmitter. Consistent with this observation, GABAA receptor activation did not stimulate MAPK cascade activation or CREB phosphorylation in mature cultures (18 days in vitro). To determine whether GABAA receptor activation during early development stimulates gene expression, we examined the inducible expression of the neurotrophin brain-derived neurotrophic factor (BDNF). Both GABA and muscimol stimulated BDNF expression, and pretreatment with U0126 attenuated GABA-induced BDNF expression. Whole cell electrophysiological recording was used to assess the effects of BDNF on GABA release. BDNF (100 ng/ml) dramatically increased the frequency of excitatory GABAergic spontaneous postsynaptic currents. Together, these data suggest a positive excitatory feedback loop between GABA and BDNF expression during early development, where GABA facilitates BDNF expression, and BDNF facilitates the synaptic release of GABA. Signaling via the MAPK cascade and the transcription factor CREB appear to play a substantial role in this process.

Download Full-text

ABCG2 C.421C>A Is Associated with Higher Trough Imatinib Plasma Levels in Patients with Chronic Myeloid Leukemia.

Blood ◽

10.1182/blood.v114.22.110.110 ◽

2009 ◽

Vol 114 (22) ◽

pp. 110-110

Author(s):

Naoto Takahashi ◽

Masatomo Miura ◽

Stuart A Scott ◽

Kenichi Sawada

Keyword(s):

Chronic Myeloid Leukemia ◽

Plasma Levels ◽

Myeloid Leukemia ◽

Drug Transport ◽

Conflicts Of Interest ◽

Nucleotide Polymorphisms ◽

Wild Type ◽

Large Patient

Abstract Abstract 110 [Background] Despite the excellent efficacy of imatinib for the treatment of chronic myeloid leukemia (CML), trough imatinib plasma levels can vary widely among patients. This may be due, in part, to inter-individual variation in imatinib metabolism and drug transport efficacy. To investigate the role of genetic variation in the pharmacokinetics of imatinib, we analyzed common single nucleotide polymorphisms within important imatinib pathway genes including ABCG2 (BCRP), ABCB1 (MDR1), ABCC2 (MRP2), CYP3A5, and SLC22A1 (OCT1) in 67 CML patients treated with imatinib. In addition, trough imatinib plasma levels were determined using high-performance liquid chromatography-tandem mass spectrometry. [Results] Distinct imatinib pharmacokinetics were identified in association with ABCG2 c.421C>A (p.Q141K; rs2231142) genotype. Specifically, the presence of the variant c.421A allele was significantly (p=0.024) associated with higher imatinib concentrations [median Cmin/Dose 2.70 (range: 1.50-8.30) ng/ml/mg; n=25] compared to patients with the wild-type ABCG2 (c.421C/C) genotype [median Cmin/Dose 2.27 (range: 0.37-5.30) ng/ml/mg; n=42]. ABCG2 is an efflux transporter for many xenobiotics, including imatinib, and is expressed at high levels in the human liver. Previous studies indicate that c.421A causes a 40% reduction in imatinib transport in vitro when compared to the wild-type genotype. Our data suggest that CML patients with ABCG2 c.421A allele may have deficient ABCG2 activity in vivo, resulting in reduced hepatic excretion of imatinib. Of note, although less common among Africans and individuals of European decent, the ABCG2 c.421C>A allele occurs at a high frequency in the Japanese (0.311) and Han Chinese (0.289) populations. [Conclusion] The association of ABCG2 c.421C>A with imatinib pharmacokinetics may explain why some Japanese CML patients administered less than 400 mg/day of imatinib have clinically sufficient trough imatinib plasma levels. Prospective studies are warranted to confirm the association between ABCG2 genotype and imatinib pharmacokinetics in large patient populations. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

OR24-01 Mutational Study of the GPR119 Receptor Binding Site

Journal of the Endocrine Society ◽

10.1210/jendso/bvaa046.1659 ◽

2020 ◽

Vol 4 (Supplement_1) ◽

Author(s):

Matthew D Rosales ◽

Frank Dean ◽

Evangelia Kotsikorou

Keyword(s):

Hydrogen Bonding ◽

Binding Site ◽

Specific Activity ◽

Md Simulations ◽

Binding Pocket ◽

Receptor Activation ◽

Activation Mechanism ◽

Receptor Model ◽

Wild Type

Abstract The GPR119 receptor, a class A G-protein coupled receptor located in the pancreatic β cells, induces insulin production when activated. Due to its specific activity, the pharmaceutical industry has identified GPR119 as a target for the treatment for type 2 diabetes. The lack of a GRP119 crystal structure has hindered the study of the receptor so our laboratory developed GPR119 active and inactive homology models. Docking studies with the inactive receptor model indicated that two leucine residues facing the binding pocket, L5.43(169) and L6.52(242), may be involved in ligand activation. Additionally, a serine at the extracellular end of the pocket, S1.32(4), may help orient of the ligand in the binding pocket via hydrogen bonding. To gain further insight into the role of these residues and the receptor activation mechanism, molecular dynamics (MD) simulations and in vitro cAMP assays of the wild type and mutant receptors were employed. The software NAMD employing the CHARMM force field was used to carry out MD simulations of the active receptor model bound with the agonist AR231453 embedded in a hydrated lipid bilayer. Preliminary results indicate that L6.52(242), located on transmembrane helix (TMH) 6, does not face directly into the binding site and does not interact with the ligand, while L5.43(169), located on TMH5, does face into the binding site, potentially interacting directly with the ligand. Also, S1.32(4), because of its extracellular location, is solvated instead of interacting with the ligand. The in vitro studies overall support the MD simulations. The mutations L6.52(242)M and L6.52(242)A appear to have minimal to no effect on agonist-induced cAMP production, compared to the wild type. In contrast, the L5.43(169)M and L5.43(169)A mutations decrease the potency of activation by AR231453, indicating that L5.43(169) changes the shape of the binding pocket, affecting ligand binding and activation. Finally, the cAMP assays show that the S1.32(4)A mutant also shows decreased activity compared to the wild type, implying that the ligand may be losing a hydrogen bonding interaction when S1.32(4) is mutated to alanine.

Download Full-text

Activating NOTCH1 Mutations Define a Distinct Subgroup of Patients With Adenoid Cystic Carcinoma Who Have Poor Prognosis, Propensity to Bone and Liver Metastasis, and Potential Responsiveness to Notch1 Inhibitors

Journal of Clinical Oncology ◽

10.1200/jco.2016.67.5264 ◽

2017 ◽

Vol 35 (3) ◽

pp. 352-360 ◽

Cited By ~ 54

Author(s):

Renata Ferrarotto ◽

Yoshitsugu Mitani ◽

Lixia Diao ◽

Irene Guijarro ◽

Jing Wang ◽

...

Keyword(s):

Bone Metastasis ◽

Poor Prognosis ◽

Regulatory Region ◽

Luciferase Reporter ◽

Wild Type ◽

Intracellular Domain ◽

Adenoid Cystic ◽

Pathway Activation ◽

Notch1 Mutations

Purpose Adenoid cystic carcinomas (ACCs) represent a heterogeneous group of chemotherapy refractory tumors, with a subset demonstrating an aggressive phenotype. We investigated the molecular underpinnings of this phenotype and assessed the Notch1 pathway as a potential therapeutic target. Methods We genotyped 102 ACCs that had available pathologic and clinical data. Notch1 activation was assessed by immunohistochemistry for Notch1 intracellular domain. Luciferase reporter assays were used to confirm Notch1 target gene expression in vitro. The Notch1 inhibitor brontictuzumab was tested in patient-derived xenografts from patients with ACC and in a patient with ACC who was enrolled in a phase I study. Results NOTCH1 mutations occurred predominantly (14 of 15 patients) in the negative regulatory region and Pro-Glu-Ser-Thr–rich domains, the same two hotspots seen in T-cell acute lymphoblastic leukemias, and led to pathway activation in vitro. NOTCH1-mutant tumors demonstrated significantly higher levels of Notch1 pathway activation than wild-type tumors on the basis of Notch1 intracellular domain staining ( P = .004). NOTCH1 mutations define a distinct aggressive ACC subgroup with a significantly higher likelihood of solid subtype ( P < .001), advanced-stage disease at diagnosis ( P = .02), higher rate of liver and bone metastasis ( P ≤ .02), shorter relapse-free survival (median, 13 v 34 months; P = .01), and shorter overall survival (median 30 v 122 months; P = .001) when compared with NOTCH1 wild-type tumors. Significant tumor growth inhibition with brontictuzumab was observed exclusively in the ACC patient-derived xenograft model that harbored a NOTCH1 activating mutation. Furthermore, an index patient with NOTCH1-mutant ACC had a partial response to brontictuzumab. Conclusion NOTCH1 mutations define a distinct disease phenotype characterized by solid histology, liver and bone metastasis, poor prognosis, and potential responsiveness to Notch1 inhibitors. Clinical studies targeting Notch1 in a genotype-defined ACC subgroup are warranted.

Download Full-text

High doses of folic acid induce a pseudo-methylenetetrahydrofolate syndrome

SAGE Open Medical Case Reports ◽

10.1177/2050313x19850435 ◽

2019 ◽

Vol 7 ◽

pp. 2050313X1985043 ◽

Cited By ~ 3

Author(s):

Dominique Cornet ◽

Arthur Clement ◽

Patrice Clement ◽

Yves Menezo

Keyword(s):

Folic Acid ◽

In Vitro Fertilization ◽

Intracytoplasmic Sperm Injection ◽

Methylenetetrahydrofolate Reductase ◽

Caucasian Woman ◽

Nucleotide Polymorphisms ◽

Wild Type ◽

Vitro Fertilization ◽

High Doses

A 41-year-old Caucasian woman with a history of infertility dating from 2011 was identified as wild-type (no mutations) for methylenetetrahydrofolate reductase single nucleotide polymorphisms (MTHFR-SNPs). Previous treatment included three failed in vitro fertilization/intracytoplasmic sperm injection cycles as well as one failed cycle of in vitro fertilization/intracytoplasmic sperm injection with donated oocytes. Counseling for a further oocyte donation cycle included advice to take high doses of folic acid (5 mG per day). Prior to initiation of this cycle, in October 2017 she attended our unit for general gynecological assessment and was found to have a slightly increased level of homocysteine, 12.2 µmol/L. A further test in February 2018 showed an increase to 17.2 µmol/L. Folic acid was stopped, and she was treated with 5-MTHF (500 µG daily), which supports the one-carbon cycle. After 5 days of treatment, her homocysteine level dropped to a baseline level of 8.2 µmol/L. As previously described in mice, high doses of folic acid can induce a “pseudo MTHFR” syndrome in wild-type patients, leading to an elevated unmetabolized folic acid syndrome which results in increased serum levels of homocysteine.

Download Full-text

MAPK pathway and B cells overactivation in multiple sclerosis revealed by phosphoproteomics and genomic analysis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1818347116 ◽

2019 ◽

Vol 116 (19) ◽

pp. 9671-9676 ◽

Cited By ~ 9

Author(s):

Ekaterina Kotelnikova ◽

Narsis A. Kiani ◽

Dimitris Messinis ◽

Inna Pertsovskaya ◽

Vicky Pliaka ◽

...

Keyword(s):

Multiple Sclerosis ◽

B Cells ◽

Kinase Inhibitors ◽

Mononuclear Cells ◽

Mapk Pathway ◽

Genomic Analysis ◽

Nucleotide Polymorphisms ◽

Peripheral Blood Mononuclear ◽

Disease Modifying Drugs

Dysregulation of signaling pathways in multiple sclerosis (MS) can be analyzed by phosphoproteomics in peripheral blood mononuclear cells (PBMCs). We performed in vitro kinetic assays on PBMCs in 195 MS patients and 60 matched controls and quantified the phosphorylation of 17 kinases using xMAP assays. Phosphoprotein levels were tested for association with genetic susceptibility by typing 112 single-nucleotide polymorphisms (SNPs) associated with MS susceptibility. We found increased phosphorylation of MP2K1 in MS patients relative to the controls. Moreover, we identified one SNP located in the PHDGH gene and another on IRF8 gene that were associated with MP2K1 phosphorylation levels, providing a first clue on how this MS risk gene may act. The analyses in patients treated with disease-modifying drugs identified the phosphorylation of each receptor’s downstream kinases. Finally, using flow cytometry, we detected in MS patients increased STAT1, STAT3, TF65, and HSPB1 phosphorylation in CD19+ cells. These findings indicate the activation of cell survival and proliferation (MAPK), and proinflammatory (STAT) pathways in the immune cells of MS patients, primarily in B cells. The changes in the activation of these kinases suggest that these pathways may represent therapeutic targets for modulation by kinase inhibitors.

Download Full-text

Mutually exclusive recurrent somatic mutations in MAP2K1 and BRAF support a central role for ERK activation in LCH pathogenesis

Blood ◽

10.1182/blood-2014-05-577825 ◽

2014 ◽

Vol 124 (19) ◽

pp. 3007-3015 ◽

Cited By ~ 219

Author(s):

Rikhia Chakraborty ◽

Oliver A. Hampton ◽

Xiaoyun Shen ◽

Stephen J. Simko ◽

Albert Shih ◽

...

Keyword(s):

Somatic Mutations ◽

Mapk Pathway ◽

Wild Type ◽

Erk Activation ◽

Mapk Kinase ◽

Erk Phosphorylation ◽

Key Points

Key Points Recurrent somatic mutations in MAP2K1 were identified in 33% of LCH lesions with wild-type BRAF. The mutant MAPK kinase 1 proteins activate ERK. The ability of MAPK pathway inhibitors to suppress MAPK kinase and ERK phosphorylation in vitro was dependent on the specific LCH mutation.

Download Full-text

Eupnea and gasping in vivo are facilitated by the activation of 5-HT2A receptors

Journal of Neurophysiology ◽

10.1152/jn.00088.2021 ◽

2021 ◽

Vol 125 (5) ◽

pp. 1543-1551

Author(s):

Kevin J. Cummings

Keyword(s):

Receptor Activation ◽

In Vitro Studies ◽

Wild Type

Previous in vitro studies suggest that 5-HT2A receptors contribute to eupnea and are necessary for fictive gasping. The current study shows that the impaired gasping displayed by neonatal TPH2−/− mice deficient in CNS serotonin is restored by a 5-HT2A receptor activation. Following 5-HT2A blockade, wild-type mice hypoventilated and their gasping resembled that of TPH2−/− mice. This study shows that both eupnea and gasping in vivo rely on the activation of 5-HT2A receptors.

Download Full-text

ERBIN deficiency links STAT3 and TGF-β pathway defects with atopy in humans

Journal of Experimental Medicine ◽

10.1084/jem.20161435 ◽

2017 ◽

Vol 214 (3) ◽

pp. 669-680 ◽

Cited By ~ 21

Author(s):

J.J. Lyons ◽

Y. Liu ◽

C.A. Ma ◽

X. Yu ◽

M.P. O’Connell ◽

...

Keyword(s):

Allergic Diseases ◽

Foxp3 Expression ◽

Receptor Activation ◽

Dominant Negative ◽

Loss Of Function ◽

Interacting Protein ◽

Naturally Occurring ◽

Pathway Activation

Nonimmunological connective tissue phenotypes in humans are common among some congenital and acquired allergic diseases. Several of these congenital disorders have been associated with either increased TGF-β activity or impaired STAT3 activation, suggesting that these pathways might intersect and that their disruption may contribute to atopy. In this study, we show that STAT3 negatively regulates TGF-β signaling via ERBB2-interacting protein (ERBIN), a SMAD anchor for receptor activation and SMAD2/3 binding protein. Individuals with dominant-negative STAT3 mutations (STAT3mut) or a loss-of-function mutation in ERBB2IP (ERBB2IPmut) have evidence of deregulated TGF-β signaling with increased regulatory T cells and total FOXP3 expression. These naturally occurring mutations, recapitulated in vitro, impair STAT3–ERBIN–SMAD2/3 complex formation and fail to constrain nuclear pSMAD2/3 in response to TGF-β. In turn, cell-intrinsic deregulation of TGF-β signaling is associated with increased functional IL-4Rα expression on naive lymphocytes and can induce expression and activation of the IL-4/IL-4Rα/GATA3 axis in vitro. These findings link increased TGF-β pathway activation in ERBB2IPmut and STAT3mut patient lymphocytes with increased T helper type 2 cytokine expression and elevated IgE.

Download Full-text

Rifampicin Transport by OATP1B1 Variants

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00955-20 ◽

2020 ◽

Vol 64 (10) ◽

Author(s):

Carlijn H. C. Litjens ◽

Jeroen J. M. W van den Heuvel ◽

Frans G. M. Russel ◽

Rob E. Aarnoutse ◽

Lindsey H. M. te Brake ◽

...

Keyword(s):

Single Nucleotide Polymorphisms ◽

Intrinsic Clearance ◽

Hek293 Cells ◽

Interindividual Variation ◽

Clinical Implications ◽

Nucleotide Polymorphisms ◽

Wild Type ◽

Single Nucleotide

ABSTRACT Single nucleotide polymorphisms in the OATP1B1 transporter have been suggested to partially explain the large interindividual variation in rifampicin exposure. HEK293 cells overexpressing wild-type (WT) or OATP1B1 variants *1b, *4, *5, and *15 were used to determine the in vitro rifampicin intrinsic clearance. For OATP1B1*5 and *15, a 36% and 42% reduction in intrinsic clearance, respectively, compared to WT was found. We consider that these differences in intrinsic clearance most likely have minor clinical implications.

Download Full-text