Structure of the phosphoinositide 3-kinase p110γ-p101 complex reveals molecular mechanism of GPCR activation

The class IB phosphoinositide 3-kinase (PI3K), PI3Kγ, is a master regulator of immune cell function, and a promising drug target for both cancer and inflammatory diseases. Critical to PI3Kγ function is the association of the p110γ catalytic subunit to either a p101 or p84 regulatory subunit, which mediates activation by G-protein coupled receptors (GPCRs). Here, we report the cryo-EM structure of a hetero-dimeric PI3Kγ complex, p110γ-p101. This structure reveals a unique assembly of catalytic and regulatory subunits that is distinct from other class I PI3K complexes. p101 mediates activation through its Gβγ binding domain, recruiting the hetero-dimer to the membrane and allowing for engagement of a secondary Gβγ binding site in p110γ. Multiple oncogenic mutations mapped to these novel interfaces and enhanced Gβγ activation. A nanobody that specifically binds to the p101-Gβγ interface blocks activation providing a novel tool to study and target p110γ-p101-specific signaling events in vivo.

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Phosphoinositide 3-Kinase Catalytic Subunit Deletion and Regulatory Subunit Deletion Have Opposite Effects on Insulin Sensitivity in Mice

Molecular and Cellular Biology ◽

10.1128/mcb.25.5.1596-1607.2005 ◽

2005 ◽

Vol 25 (5) ◽

pp. 1596-1607 ◽

Cited By ~ 114

Author(s):

Saskia M. Brachmann ◽

Kohjiro Ueki ◽

Jeffrey A. Engelman ◽

Ronald C. Kahn ◽

Lewis C. Cantley

Keyword(s):

Insulin Sensitivity ◽

Glucose Uptake ◽

Mammalian Cells ◽

Ex Vivo ◽

Regulatory Subunit ◽

Regulatory Subunits ◽

Pi3k Activation ◽

Insulin Dependent ◽

Phosphoinositide 3 Kinase

ABSTRACT Studies ex vivo have shown that phosphoinositide 3-kinase (PI3K) activity is necessary but not sufficient for insulin-stimulated glucose uptake. Unexpectedly, mice lacking either of the PI3K regulatory subunits p85α or p85β exhibit increased insulin sensitivity. The insulin hypersensitivity is particularly unexpected in p85α−/− p55α−/− p50α−/− mice, where a decrease in p110α and p110β catalytic subunits was observed in insulin-sensitive tissues. These results raised the possibility that decreasing total PI3K available for stimulation by insulin might circumvent negative feedback loops that ultimately shut off insulin-dependent glucose uptake in vivo. Here we present results arguing against this explanation. We show that p110α+/− p110β+/− mice exhibit mild glucose intolerance and hyperinsulinemia in the fasted state. Unexpectedly, p110α+/− p110β+/− mice showed a ∼50% decrease in p85 expression in liver and muscle. Consistent with this in vivo observation, knockdown of p110 by RNA interference in mammalian cells resulted in loss of p85 proteins due to decreased protein stability. We propose that insulin sensitivity is regulated by a delicate balance between p85 and p110 subunits and that p85 subunits mediate a negative role in insulin signaling independent of their role as mediators of PI3K activation.

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PI3Kβ—A Versatile Transducer for GPCR, RTK, and Small GTPase Signaling

Endocrinology ◽

10.1210/en.2018-00843 ◽

2019 ◽

Vol 160 (3) ◽

pp. 536-555 ◽

Cited By ~ 10

Author(s):

Anne R Bresnick ◽

Jonathan M Backer

Keyword(s):

G Proteins ◽

Tyrosine Kinases ◽

Small Gtpases ◽

Small Gtpase ◽

Catalytic Subunits ◽

Regulatory Subunits ◽

Phosphoinositide 3 Kinase ◽

G Protein Coupled

AbstractThe phosphoinositide 3-kinase (PI3K) family includes eight distinct catalytic subunits and seven regulatory subunits. Only two PI3Ks are directly regulated downstream from G protein–coupled receptors (GPCRs): the class I enzymes PI3Kβ and PI3Kγ. Both enzymes produce phosphatidylinositol 3,4,5-trisposphate in vivo and are regulated by both heterotrimeric G proteins and small GTPases from the Ras or Rho families. However, PI3Kβ is also regulated by direct interactions with receptor tyrosine kinases (RTKs) and their tyrosine phosphorylated substrates, and similar to the class II and III PI3Ks, it binds activated Rab5. The unusually complex regulation of PI3Kβ by small and trimeric G proteins and RTKs leads to a rich landscape of signaling responses at the cellular and organismic levels. This review focuses first on the regulation of PI3Kβ activity in vitro and in cells, and then summarizes the biology of PI3Kβ signaling in distinct tissues and in human disease.

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T-cell function is partially maintained in the absence of class IA phosphoinositide 3-kinase signaling

Blood ◽

10.1182/blood-2006-07-038620 ◽

2006 ◽

Vol 109 (7) ◽

pp. 2894-2902 ◽

Cited By ~ 40

Author(s):

Jonathan A. Deane ◽

Michael G. Kharas ◽

Jean S. Oak ◽

Linda N. Stiles ◽

Ji Luo ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

Cell Function ◽

Pi3k Signaling ◽

Regulatory Subunits ◽

Cell Functions ◽

Phosphoinositide 3 Kinase

Abstract The class IA subgroup of phosphoinositide 3-kinase (PI3K) is activated downstream of antigen receptors, costimulatory molecules, and cytokine receptors on lymphocytes. Targeted deletion of individual genes for class IA regulatory subunits severely impairs the development and function of B cells but not T cells. Here we analyze conditional mutant mice in which thymocytes and T cells lack the major class IA regulatory subunits p85α, p55α, p50α, and p85β. These cells exhibit nearly complete loss of PI3K signaling downstream of the T-cell receptor (TCR) and CD28. Nevertheless, T-cell development is largely unperturbed, and peripheral T cells show only partial impairments in proliferation and cytokine production in vitro. Both genetic and pharmacologic experiments suggest that class IA PI3K signaling plays a limited role in T-cell proliferation driven by TCR/CD28 clustering. In vivo, class IA–deficient T cells provide reduced help to B cells but show normal ability to mediate antiviral immunity. Together these findings provide definitive evidence that class IA PI3K regulatory subunits are essential for a subset of T-cell functions while challenging the notion that this signaling mechanism is a critical mediator of costimulatory signals downstream of CD28.

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Erratum: Bisphosphonate-induced differential modulation of immune cell function in gingiva and bone marrow in vivo: Role in osteoclast-mediated NK cell activation

Oncotarget ◽

10.18632/oncotarget.6431 ◽

2015 ◽

Vol 6 (38) ◽

pp. 41398-41398 ◽

Cited By ~ 1

Author(s):

Han-Ching Tseng ◽

Keiichi Kanayama ◽

Kawaljit Kaur ◽

So-Hyun Park ◽

Sil Park ◽

...

Keyword(s):

Bone Marrow ◽

Cell Activation ◽

Cell Function ◽

Immune Cell ◽

Nk Cell ◽

Differential Modulation ◽

Immune Cell Function

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Prospects for use of peptides and their derivatives, structurally corresponding to the G protein-coupled receptors, in medicine

Biomeditsinskaya Khimiya ◽

10.18097/pbmc20156101019 ◽

2015 ◽

Vol 61 (1) ◽

pp. 19-29 ◽

Cited By ~ 1

Author(s):

A.O. Shpakov ◽

E.A. Shpakova

Keyword(s):

G Protein ◽

Intracellular Signaling ◽

High Efficiency ◽

Clinical Medicine ◽

Inflammatory Diseases ◽

G Protein Coupled Receptors ◽

Signaling Cascades ◽

G Protein Coupled

The regulation of signaling pathways involved in the control of many physiological functions is carried out via the heterotrimeric G protein-coupled receptors (GPCR). The search of effective and selective regulators of GPCR and intracellular signaling cascades coupled with them is one of the important problems of modern fundamental and clinical medicine. Recently data suggest that synthetic peptides and their derivatives, structurally corresponding to the intracellular and transmembrane regions of GPCR, can interact with high efficiency and selectivity with homologous receptors and influence, thus, the functional activity of intracellular signaling cascades and fundamental cellular processes controlled by them. GPCR-peptides are active in both in vitro and in vivo. They regulate hematopoiesis, angiogenesis and cell proliferation, inhibit tumor growth and metastasis, and prevent the inflammatory diseases and septic shock. These data show greatest prospects in the development of the new generations of drugs based on GPCR-derived peptides, capable of regulating the important functions of the organism.

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Administration of cardiac mesenchymal cells modulates innate immunity in the acute phase of myocardial infarction in mice

Scientific Reports ◽

10.1038/s41598-020-71580-z ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Yi Kang ◽

Marjan Nasr ◽

Yiru Guo ◽

Shizuka Uchida ◽

Tyler Weirick ◽

...

Keyword(s):

Myocardial Infarction ◽

Innate Immunity ◽

Mechanism Of Action ◽

Cell Activation ◽

Cell Function ◽

Immune Cell ◽

Mesenchymal Cell ◽

Cell Types

Abstract Although cardiac mesenchymal cell (CMC) therapy mitigates post-infarct cardiac dysfunction, the underlying mechanisms remain unidentified. It is acknowledged that donor cells are neither appreciably retained nor meaningfully contribute to tissue regeneration—suggesting a paracrine-mediated mechanism of action. As the immune system is inextricably linked to wound healing/remodeling in the ischemically injured heart, the reparative actions of CMCs may be attributed to their immunoregulatory properties. The current study evaluated the consequences of CMC administration on post myocardial infarction (MI) immune responses in vivo and paracrine-mediated immune cell function in vitro. CMC administration preferentially elicited the recruitment of cell types associated with innate immunity (e.g., monocytes/macrophages and neutrophils). CMC paracrine signaling assays revealed enhancement in innate immune cell chemoattraction, survival, and phagocytosis, and diminished pro-inflammatory immune cell activation; data that identifies and catalogues fundamental immunomodulatory properties of CMCs, which have broad implications regarding the mechanism of action of CMCs in cardiac repair.

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Critical Analysis of Non-Thermal Plasma-Driven Modulation of Immune Cells from Clinical Perspective

International Journal of Molecular Sciences ◽

10.3390/ijms21176226 ◽

2020 ◽

Vol 21 (17) ◽

pp. 6226 ◽

Cited By ~ 1

Author(s):

Barbora Smolková ◽

Adam Frtús ◽

Mariia Uzhytchak ◽

Mariia Lunova ◽

Šárka Kubinová ◽

...

Keyword(s):

Immune Cells ◽

Thermal Plasma ◽

Critical Analysis ◽

Cell Function ◽

Immune Cell ◽

Real Life ◽

Research Strategy ◽

Cellular Mechanisms ◽

Non Thermal Plasma

The emerged field of non-thermal plasma (NTP) shows great potential in the alteration of cell redox status, which can be utilized as a promising therapeutic implication. In recent years, the NTP field considerably progresses in the modulation of immune cell function leading to promising in vivo results. In fact, understanding the underlying cellular mechanisms triggered by NTP remains incomplete. In order to boost the field closer to real-life clinical applications, there is a need for a critical overview of the current state-of-the-art. In this review, we conduct a critical analysis of the NTP-triggered modulation of immune cells. Importantly, we analyze pitfalls in the field and identify persisting challenges. We show that the identification of misconceptions opens a door to the development of a research strategy to overcome these limitations. Finally, we propose the idea that solving problems highlighted in this review will accelerate the clinical translation of NTP-based treatments.

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IMMU-46. EXAMINATION OF THE EFFECTS OF DEXAMETHASONE ON THE EFFICACY OF IMMUNOTHERAPY IN GLIOMA USING GENE-MEDIATED CYTOTOXIC IMMUNOTHERAPY

Neuro-Oncology ◽

10.1093/neuonc/noz175.538 ◽

2019 ◽

Vol 21 (Supplement_6) ◽

pp. vi129-vi129

Author(s):

Marilin Koch ◽

Mykola Zdioruk ◽

M Oskar Nowicki ◽

Estuardo Aguilar ◽

Laura Aguilar ◽

...

Keyword(s):

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Cell Function ◽

Immune Cell ◽

Symptomatic Treatment ◽

Tumor Cell Death ◽

The Impact

Abstract RATIONALE Dexamethasone is frequently used in symptomatic treatment of glioma patients, although it is known to cause immune suppression. Checkpoint inhibitor immunotherapies have not yet been successful in glioma treatments. Gene-mediated cytotoxic immunotherapy (GMCI) is an immunotherapeutic approach that uses aglatimagene besadenovec with an anti-herpetic prodrug to induce immunogenic tumor cell death and immune cell attraction to the tumor site with potent CD8 T cell activation. GMCI is currently in clinical trials for solid tumors including glioblastoma, where it showed encouraging survival results in a Phase 2 study that did not limit the use of dexamethasone. However, the effects of dexamethasone on its efficacy have not been explored. METHODS We investigated the effects of dexamethasone on GMCI in vitro using cytotoxicity and T-cell-killing assays in glioblastoma cell lines. The impact of dexamethasone in vivo was assessed in an orthotopic syngeneic murine glioblastoma model. RESULTS Cyotoxicity assays showed that Dexamethasone has a slight impact on GMCI in vitro. In contrast, we observed a highly significant effect in T-cell-functional assays in which killing was greatly impaired. Immune cell response assays revealed a reduced T-cell proliferation after co-culture with supernatant from dexamethasone or combination treated glioblastoma cells in contrast to GMCI alone. In a murine model, the combination of GMCI and dexamethasone resulted in a significant reduction in median symptom-free survival (29d) in comparison to GMCI alone (39.5d) (P = 0.0184). CONCLUSION Our data suggest that high doses of dexamethasone may negatively impact the efficacy of immunotherapy for glioma, which may be a consequence of impaired T cell function. These results support the idea that there is a need in identifying possible alternatives to dexamethasone to maximize the effectiveness of immunostimulatory therapies such as GMCI.

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Two-Photon Imaging of Immune Cell Dynamics: Moving from 2D Fixed Tissue Sections to 3D Dynamic Histology In Vivo

Blood ◽

10.1182/blood.v122.21.sci-24.sci-24 ◽

2013 ◽

Vol 122 (21) ◽

pp. SCI-24-SCI-24

Author(s):

Mark J. Miller

Keyword(s):

Immune Responses ◽

Bacterial Infection ◽

Reperfusion Injury ◽

Immune Cell ◽

Ischemia Reperfusion Injury ◽

Inflammatory Diseases ◽

Ischemia Reperfusion ◽

Neutrophil Recruitment ◽

Neutrophil Extravasation

Abstract Cell-mediated immune responses are highly dependent on environmental context, thus making in vivo studies an important complement to in vitro and molecular approaches. Two-photon microscopy (2PM) is a fluorescence based imaging approach that allows single-cell dynamics to be studied directly in their 3D native tissue context. 2PM is an ideal approach for analyzing leukocyte trafficking dynamics quantitatively and testing cellular immune mechanisms in vivo. Several example applications will be presented where 2PM has uncovered novel immunological phenomena and provided fresh insight into immune responses to infection, autoimmunity and cancer. While 2P imaging has been used extensively to study immune cell trafficking and function in mice, progress is being made to use this imaging technique on clinical biopsy specimens to acquire a multi-dimensional picture of human tissue pathology. We used in vivo 2PM in pre-clinical models of arthritis and bacterial infection to compare and contrast the role of monocytes on neutrophil recruitment. The rapid recruitment of neutrophils and monocytes is critical to early host immune responses to bacterial infection. However, leukocyte recruitment also contributes to chronic inflammatory diseases such as human rheumatoid arthritis. Understanding how cell recruitment is regulated in different inflammatory contexts is crucial for developing safe and effective anti-inflammatory therapies. We found that monocyte depletion with clodronate-liposomes prevented arthritis development in a modified K/BxN serum transfer arthritis model. This protective effect was associated with significantly reduced neutrophil transendothelial migration efficiency. Furthermore, single-cell tracking of a minor population of extravasated neutrophils showed that neutrophil migration and chemotaxis in interstitial tissues was disrupted, contributing to decreased cell localization at phalangeal joints. Similar results were obtained when CCR2+ monocytes were depleted selectively using the monoclonal antibody MC-21, thus implicating CCR2+ monocytes as key regulators of neutrophil extravasation during arthritis initiation. In contrast, neutrophil recruitment to subcutaneous bacterial challenge remained intact and neutrophil extravasation and chemotaxis to sites of infection was not significantly different as compared to non-depleted controls. We also examined whether neutrophil extravasation during acute pulmonary inflammation required monocytes. Neutrophil recruitment in vivo was assessed in a mouse lung transplant-mediated ischemia reperfusion injury model. Similar to the results in the arthritis model, neutrophil recruitment in response to ischemia reperfusion injury was also monocyte dependent. In addition, Ccr2 knockout recipient mice were protected for ischemia reperfusion injury. Results from these complementary mouse models implicate CCR2+ monocytes as key regulators of neutrophil extravasation and chemotaxis in under conditions of aseptic inflammation and further suggest that the cell recruitment signals that that operate during bacterial infection may be quantitatively and/or qualitatively distinct. These studies raise the intriguing possibility that targeting monocytes during chronic inflammatory diseases such as rheumatoid arthritis or acute inflammatory conditions such as ischemia reperfusion injury might provide safer and more selective anti-inflammatory therapies than those that target neutrophils directly. Disclosures: No relevant conflicts of interest to declare.

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The P2Y6 Receptor Stimulates Bone Resorption by Osteoclasts

Endocrinology ◽

10.1210/en.2011-1073 ◽

2011 ◽

Vol 152 (10) ◽

pp. 3706-3716 ◽

Cited By ~ 22

Author(s):

Isabel R. Orriss ◽

Ning Wang ◽

Geoffrey Burnstock ◽

Timothy R. Arnett ◽

Alison Gartland ◽

...

Keyword(s):

Cell Function ◽

Bone Cell ◽

Extracellular Nucleotides ◽

P2y6 Receptor ◽

Tomographic Analysis ◽

G Protein Coupled ◽

Resorptive Activity

Accumulating evidence indicates that extracellular nucleotides, signaling through P2 receptors, play a significant role in bone remodeling. Osteoclasts (the bone-resorbing cell) and osteoblasts (the bone-forming cell) display expression of the G protein-coupled P2Y6 receptor, but the role of this receptor in modulating cell function is unclear. Here, we demonstrate that extracellular UDP, acting via P2Y6 receptors, stimulates the formation of osteoclasts from precursor cells, while also enhancing the resorptive activity of mature osteoclasts. Furthermore, osteoclasts derived from P2Y6 receptor-deficient (P2Y6R−/−) animals displayed defective function in vitro. Using dual energy x-ray absorptiometry scanning and microcomputed tomographic analysis we showed that P2Y6R−/− mice have increased bone mineral content, cortical bone volume, and cortical thickness in the long bones and spine, whereas trabecular bone parameters were unaffected. Histomorphometric analysis showed the perimeter of the bone occupied by osteoclasts on the endocortical and trabecular surfaces was decreased in P2Y6R−/− mice. Taken together these results show the P2Y6 receptor may play an important role in the regulation of bone cell function in vivo.

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