Faculty Opinions recommendation of Structure of the phosphoinositide 3-kinase (PI3K) p110γ-p101 complex reveals molecular mechanism of GPCR activation.

Most chemoattractants rely on activation of the heterotrimeric G-protein Gαi to regulate directional cell migration, but few links from Gαi to chemotactic effectors are known. Through affinity chromatography using primary neutrophil lysate, we identify Homer3 as a novel Gαi2-binding protein. RNA interference–mediated knockdown of Homer3 in neutrophil-like HL-60 cells impairs chemotaxis and the establishment of polarity of phosphatidylinositol 3,4,5-triphosphate (PIP3) and the actin cytoskeleton, as well as the persistence of the WAVE2 complex. Most previously characterized proteins that are required for cell polarity are needed for actin assembly or activation of core chemotactic effectors such as the Rac GTPase. In contrast, Homer3-knockdown cells show normal magnitude and kinetics of chemoattractant-induced activation of phosphoinositide 3-kinase and Rac effectors. Chemoattractant-stimulated Homer3-knockdown cells also exhibit a normal initial magnitude of actin polymerization but fail to polarize actin assembly and intracellular PIP3 and are defective in the initiation of cell polarity and motility. Our data suggest that Homer3 acts as a scaffold that spatially organizes actin assembly to support neutrophil polarity and motility downstream of GPCR activation.

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

10.1101/2021.06.01.446612 ◽

2021 ◽

Author(s):

Manoj K Rathinaswamy ◽

Udit Dalwadi ◽

Kaelin D Fleming ◽

Carson Adams ◽

Jordan TB Stariha ◽

...

Keyword(s):

Cell Function ◽

Immune Cell ◽

Inflammatory Diseases ◽

Regulatory Subunit ◽

Regulatory Subunits ◽

Gpcr Activation ◽

Oncogenic Mutations ◽

Phosphoinositide 3 Kinase ◽

G Protein Coupled

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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Noncanonical regulation of insulin-mediated ERK activation by phosphoinositide 3-kinase γ

Molecular Biology of the Cell ◽

10.1091/mbc.e16-12-0864 ◽

2017 ◽

Vol 28 (22) ◽

pp. 3112-3122 ◽

Cited By ~ 4

Author(s):

Maradumane L. Mohan ◽

Arunachal Chatterjee ◽

Swetha Ganapathy ◽

Sromona Mukherjee ◽

Sowmya Srikanthan ◽

...

Keyword(s):

Cardiac Fibroblasts ◽

Knock Out ◽

Erk Activation ◽

Cellular Processes ◽

Gpcr Activation ◽

Extracellular Signal ◽

Phosphatase 2A ◽

Epidermal Growth ◽

Phosphoinositide 3 Kinase ◽

Knock Out Mice

Classically Class IB phosphoinositide 3-kinase (PI3Kγ) plays a role in extracellular signal–regulated kinase (ERK) activation following G-protein coupled receptor (GPCR) activation. Knock-down of PI3Kγ unexpectedly resulted in loss of ERK activation to receptor tyrosine kinase agonists such as epidermal growth factor or insulin. Mouse embryonic fibroblasts (MEFs) or primary adult cardiac fibroblasts isolated from PI3Kγ knock-out mice (PI3KγKO) showed decreased insulin-stimulated ERK activation. However, expression of kinase-dead PI3Kγ resulted in rescue of insulin-stimulated ERK activation. Mechanistically, PI3Kγ sequesters protein phosphatase 2A (PP2A), disrupting ERK–PP2A interaction, as evidenced by increased ERK–PP2A interaction and associated PP2A activity in PI3KγKO MEFs, resulting in decreased ERK activation. Furthermore, β-blocker carvedilol-mediated β-arrestin-dependent ERK activation is significantly reduced in PI3KγKO MEF, suggesting accelerated dephosphorylation. Thus, instead of classically mediating the kinase arm, PI3Kγ inhibits PP2A by scaffolding and sequestering, playing a key parallel synergistic step in sustaining the function of ERK, a nodal enzyme in multiple cellular processes.

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HDX-MS optimized approach to characterize nanobodies as tools for biochemical and structural studies of class IB phosphoinositide 3-kinases

10.1101/2021.06.01.446614 ◽

2021 ◽

Author(s):

Manoj K Rathinaswamy ◽

Kaelin D Fleming ◽

Udit Dalwadi ◽

Els Pardon ◽

Noah J Harris ◽

...

Keyword(s):

Signaling Pathways ◽

Pi3k Pathway ◽

Regulatory Subunit ◽

Single Chain ◽

Lipid Kinase ◽

Exchange Mass Spectrometry ◽

Gpcr Activation ◽

Phosphoinositide 3 Kinase ◽

Hdx Ms ◽

Class Ib

There is considerable interest in developing antibodies as modulators of signaling pathways. One of the most important signaling pathways in higher eukaryotes is the phosphoinositide 3-kinase (PI3K) pathway, which plays fundamental roles in growth, metabolism and immunity. The class IB PI3K, PI3Kγ, is a heterodimeric complex composed of a catalytic p110γ subunit bound to a p101 or p84 regulatory subunit. PI3Kγ is a critical component in multiple immune signaling processes and is dependent on activation by Ras and GPCRs to mediate its cellular roles. Here we describe the rapid and efficient characterization of multiple PI3K single chain camelid nanobodies using hydrogen deuterium exchange mass spectrometry (HDX-MS) for structural and biochemical studies. This allowed us to identify nanobodies that stimulated lipid kinase activity, blocked Ras activation and specifically inhibited p101-mediated GPCR activation. Overall, this reveals novel insight into PI3Kγ regulation and identifies sites that may be exploited for therapeutic development.

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Phosphoinositide 3-kinase regulates β2-adrenergic receptor endocytosis by AP-2 recruitment to the receptor/β-arrestin complex

The Journal of Cell Biology ◽

10.1083/jcb.200202113 ◽

2002 ◽

Vol 158 (3) ◽

pp. 563-575 ◽

Cited By ~ 134

Author(s):

Sathyamangla V. Naga Prasad ◽

Stéphane A. Laporte ◽

Dean Chamberlain ◽

Marc G. Caron ◽

Larry Barak ◽

...

Keyword(s):

Molecular Mechanism ◽

Adaptor Protein ◽

Receptor Internalization ◽

Coated Pits ◽

Receptor Endocytosis ◽

Sequential Binding ◽

Β2 Adrenergic Receptor ◽

Clathrin Adaptor ◽

Phosphoinositide 3 Kinase ◽

Protein Recruitment

Internalization of β-adrenergic receptors (βARs) occurs by the sequential binding of β-arrestin, the clathrin adaptor AP-2, and clathrin. D-3 phosphoinositides, generated by the action of phosphoinositide 3-kinase (PI3K) may regulate the endocytic process; however, the precise molecular mechanism is unknown. Here we demonstrate that βARKinase1 directly interacts with the PIK domain of PI3K to form a cytosolic complex. Overexpression of the PIK domain displaces endogenous PI3K from βARK1 and prevents βARK1-mediated translocation of PI3K to activated β2ARs. Furthermore, disruption of the βARK1/PI3K interaction inhibits agonist-stimulated AP-2 adaptor protein recruitment to the β2AR and receptor endocytosis without affecting the internalization of other clathrin dependent processes such as internalization of the transferrin receptor. In contrast, AP-2 recruitment is enhanced in the presence of D-3 phospholipids, and receptor internalization is blocked in presence of the specific phosphatidylinositol-3,4,5-trisphosphate lipid phosphatase PTEN. These findings provide a molecular mechanism for the agonist-dependent recruitment of PI3K to βARs, and support a role for the localized generation of D-3 phosphoinositides in regulating the recruitment of the receptor/cargo to clathrin-coated pits.

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Antidiabetic Effects of a Chinese Herbal Medicinal Compound Sangguayin Preparation via PI3K/Akt Signaling Pathway in db/db Mice

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2018/2010423 ◽

2018 ◽

Vol 2018 ◽

pp. 1-11 ◽

Cited By ~ 2

Author(s):

Qichang Xing ◽

Yunzhong Chen

Keyword(s):

Skeletal Muscle ◽

Insulin Receptor ◽

Molecular Mechanism ◽

Glucose Transporter ◽

Blood Lipid ◽

Akt Signaling ◽

Chinese Herbs ◽

Antidiabetic Activity ◽

Long Time ◽

Phosphoinositide 3 Kinase

Sangguayin (SGY), comprising four types of Chinese herbs, can be used as both food and medicine and has been clinically used to treat type 2 diabetes mellitus (T2DM) for a long time. Our previous study demonstrated the antidiabetic effect of SGY in experimental T2DM rats fed with a high-fat diet and treated with a low dose of streptozotocin. However, its mechanism of action is questionable. In this study, we refined the traditional SGY decoction and investigated its antidiabetic activity in db/db mice. We evaluated the possible molecular mechanism using skeletal muscle tissues. The results show that the treatment with SGY preparation resulted in a decrease in the blood glucose, glycated serum protein, and blood lipid levels and an improvement in the glucose tolerance as well as insulin resistance. In addition, SGY preparation remarkably upregulated the expression of insulin receptor, insulin receptor substrate-1, phosphoinositide 3 kinase (PI3K), protein kinase B (Akt), and glucose transporter type 4 (GLUT4). Thus, SGY preparation is an effective agent for the treatment of T2DM, and its molecular mechanism may be related to the regulation of PI3K/Akt signaling in the skeletal muscle.

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