Endosomal cAMP production broadly impacts the cellular phosphoproteome

AbstractEndosomal signaling from G protein-coupled receptors (GPCRs) has emerged as a novel paradigm with important pharmacological and physiological implications. Yet, our knowledge of the functional consequences of activating intracellular GPCRs is incomplete. To address this gap, we combined an optogenetic approach for site-specific generation of the prototypical second messenger cyclic AMP (cAMP) with unbiased mass spectrometry-based analysis of phosphoproteomic effects. We identified 218 unique, high-confidence sites whose phosphorylation is either increased or decreased in response to cAMP production. We next determined that cAMP produced from endosomes led to more robust changes in phosphorylation than cAMP produced from the plasma membrane. Remarkably, this was true for the entire repertoire of identified targets, and irrespective of their annotated sub-cellular localization. Furthermore, we identified a particularly strong endosome bias for a subset of proteins that are dephosphorylated in response to cAMP. Through bioinformatics analysis, we established these targets as putative substrates for protein phosphatase 2A (PP2A), and we propose compartmentalized activation of PP2A-B56δ as the likely underlying mechanism. Altogether, our study extends the concept that endosomal signaling is a significant functional contributor to cellular responsiveness by establishing a unique role for localized cAMP production in defining categorically distinct phosphoresponses.

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The tyrosine phosphatase CD148 is an essential positive regulator of platelet activation and thrombosis

Blood ◽

10.1182/blood-2008-08-174318 ◽

2009 ◽

Vol 113 (20) ◽

pp. 4942-4954 ◽

Cited By ~ 84

Author(s):

Yotis A. Senis ◽

Michael G. Tomlinson ◽

Stuart Ellison ◽

Alexandra Mazharian ◽

Jenson Lim ◽

...

Keyword(s):

G Protein ◽

Platelet Activation ◽

Drug Target ◽

Tyrosine Phosphatase ◽

Underlying Mechanism ◽

G Protein Coupled Receptors ◽

Bleeding Tendency ◽

Platelet Surface ◽

Surface Receptors ◽

G Protein Coupled

Abstract Platelets play a fundamental role in hemostasis and thrombosis. They are also involved in pathologic conditions resulting from blocked blood vessels, including myocardial infarction and ischemic stroke. Platelet adhesion, activation, and aggregation at sites of vascular injury are regulated by a diverse repertoire of tyrosine kinase–linked and G protein–coupled receptors. Src family kinases (SFKs) play a central role in initiating and propagating signaling from several platelet surface receptors; however, the underlying mechanism of how SFK activity is regulated in platelets remains unclear. CD148 is the only receptor-like protein tyrosine phosphatase identified in platelets to date. In the present study, we show that mutant mice lacking CD148 exhibited a bleeding tendency and defective arterial thrombosis. Basal SFK activity was found to be markedly reduced in CD148-deficient platelets, resulting in a global hyporesponsiveness to agonists that signal through SFKs, including collagen and fibrinogen. G protein–coupled receptor responses to thrombin and other agonists were also marginally reduced. These results highlight CD148 as a global regulator of platelet activation and a novel antithrombotic drug target.

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BRET Analysis of GPCR Dimers in Neurons and Non-Neuronal Cells: Evidence for Inactive, Agonist, and Constitutive Conformations

International Journal of Molecular Sciences ◽

10.3390/ijms221910638 ◽

2021 ◽

Vol 22 (19) ◽

pp. 10638

Author(s):

Chayma El Khamlichi ◽

Laetitia Cobret ◽

Jean-Michel Arrang ◽

Séverine Morisset-Lopez

Keyword(s):

Cortical Neurons ◽

G Protein Coupled Receptors ◽

Constitutive Activity ◽

Western Blots ◽

Inverse Agonists ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Functional Consequences ◽

G Protein Coupled

G-protein-coupled receptors (GPCRs) are dimeric proteins, but the functional consequences of the process are still debated. Active GPCR conformations are promoted either by agonists or constitutive activity. Inverse agonists decrease constitutive activity by promoting inactive conformations. The histamine H3 receptor (H3R) is the target of choice for the study of GPCRs because it displays high constitutive activity. Here, we study the dimerization of recombinant and brain H3R and explore the effects of H3R ligands of different intrinsic efficacy on dimerization. Co-immunoprecipitations and Western blots showed that H3R dimers co-exist with monomers in transfected HEK 293 cells and in rodent brains. Bioluminescence energy transfer (BRET) analysis confirmed the existence of spontaneous H3R dimers, not only in living HEK 293 cells but also in transfected cortical neurons. In both cells, agonists and constitutive activity of the H3R decreased BRET signals, whereas inverse agonists and GTPγS, which promote inactive conformations, increased BRET signals. These findings show the existence of spontaneous H3R dimers not only in heterologous systems but also in native tissues, which are able to adopt a number of allosteric conformations, from more inactive to more active states.

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Glucagon counteracts interleukin-6-dependent gene expression by redundant action of Epac and PKA

Biological Chemistry ◽

10.1515/bc.2011.171 ◽

2011 ◽

Vol 392 (12) ◽

pp. 1123-1134 ◽

Cited By ~ 8

Author(s):

Christina Khouri ◽

Anna Dittrich ◽

Sara Dutton Sackett ◽

Bernd Denecke ◽

Christian Trautwein ◽

...

Keyword(s):

Gene Expression ◽

G Protein ◽

Acute Phase ◽

Interleukin 6 ◽

Biological Response ◽

Underlying Mechanism ◽

G Protein Coupled Receptors ◽

Counterregulatory Hormones ◽

The Impact ◽

G Protein Coupled

AbstractInflammation is the biological response to injurious stimuli. In the initial phase of the inflammatory process, interleukin-6 (IL-6) is the main inducer of acute phase protein expression in the liver. A prolonged acute phase response is characterised by a disturbed glucose homeostasis and elevated levels of IL-6, insulin, and counterregulatory hormones such as glucagon. Several studies deal with the impact of IL-6 on glucagon-dependent gene expression. In contrast, only very little is known about the influence of G-protein-coupled receptors on IL-6 signalling. Therefore, the aim of this study is to elucidate the regulation of IL-6-induced gene expression by glucagon. We could reveal a novel mechanism of negative regulation of IL-6-induced MAP kinase activation by glucagon in primary murine hepatocytes. IL-6-dependent induction of the ERK-dependent target geneTfpi2, coding for a Kunitz-type serine protease inhibitor, was strongly down-regulated by glucagon treatment. Studying the underlying mechanism revealed a redundant action of the signalling molecules exchange protein activated by cyclic AMP (Epac) and protein kinase A. The metabolic hormone glucagon interferes in IL-6-induced gene expression. This observation is indicative for a regulatory role of G-protein-coupled receptors in the IL-6-dependent inflammatory response.

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Selectivity and functional consequences of interactions of family A G protein-coupled receptors with neurochondrin and periplakin

Journal of Neurochemistry ◽

10.1111/j.1471-4159.2009.05918.x ◽

2009 ◽

Vol 109 (1) ◽

pp. 182-192 ◽

Cited By ~ 12

Author(s):

Richard J. Ward ◽

Laura Jenkins ◽

Graeme Milligan

Keyword(s):

G Protein ◽

G Protein Coupled Receptors ◽

Functional Consequences ◽

G Protein Coupled

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G Protein-coupled Receptors in Cancer Stem Cells

Current Pharmaceutical Design ◽

10.2174/1381612826666200305130009 ◽

2020 ◽

Vol 26 (17) ◽

pp. 1952-1963 ◽

Cited By ~ 1

Author(s):

Yuhong Jiang ◽

Xin Zhuo ◽

Canquan Mao

Keyword(s):

Stem Cells ◽

Cancer Therapy ◽

Cancer Stem Cells ◽

G Protein ◽

Drug Targets ◽

Underlying Mechanism ◽

G Protein Coupled Receptors ◽

Cell Functions ◽

Exogenous Ligands ◽

G Protein Coupled

G protein-coupled receptors (GPCRs) are highly expressed on a variety of tumour tissues while several GPCR exogenous ligands become marketed pharmaceuticals. In recent decades, cancer stem cells (CSCs) become widely investigated drug targets for cancer therapy but the underlying mechanism is still not fully elucidated. There are vigorous participations of GPCRs in CSCs-related signalling and functions, such as biomarkers for CSCs, activation of Wnt, Hedgehog (HH) and other signalling to facilitate CSCs progressions. This relationship can not only uncover a novel molecular mechanism for GPCR-mediated cancer cell functions but also assist our understanding of maintaining and modulating CSCs. Moreover, GPCR antagonists and monoclonal antibodies could be applied to impair CSCs functions and consequently attenuate tumour growth, some of which have been undergoing clinical studies and are anticipated to turn into marketed anticancer drugs. Therefore, this review summarizes and provides sufficient evidences on the regulation of GPCR signalling in the maintenance, differentiation and pluripotency of CSCs, suggesting that targeting GPCRs on the surface of CSCs could be potential therapeutic strategies for cancer therapy.

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Genetically encoded intrabody sensors illuminate structural and functional diversity in GPCR-β-arrestin complexes

10.1101/651463 ◽

2019 ◽

Author(s):

Mithu Baidya ◽

Punita Kumari ◽

Hemlata Dwivedi ◽

Eshan Ghosh ◽

Badr Sokrat ◽

...

Keyword(s):

Underlying Mechanism ◽

G Protein Coupled Receptors ◽

Interaction Patterns ◽

Functional Roles ◽

Functional Differences ◽

Conformational Diversity ◽

Cellular Context ◽

Spatio Temporal ◽

G Protein Coupled ◽

Fine Tune

AbstractInteraction of β-arrestins (βarrs) upon agonist-stimulation is a hallmark of G protein-coupled receptors (GPCRs) resulting in receptor desensitization, endocytosis and signaling. Although overall functional roles of βarrs are typically believed to be conserved across different receptors, emerging data now clearly unveils receptor-specific functional contribution of βarrs. The underlying mechanism however remains mostly speculative and represents a key missing link in our current understanding of GPCR signaling and regulatory paradigms. Here, we develop synthetic intrabody-based conformational sensors that help us visualize the assembly and trafficking of GPCR-βarr1 complexes in cellular context for a broad set of receptors with spatio-temporal resolution. Surprisingly, these conformational sensors reveal a previously unappreciated level of diversity in GPCR-βarr complexes that extends beyond the current framework of affinity-based classification and phosphorylation-code-based interaction patterns. More importantly, this conformational diversity arising from spatial signature of phosphorylation sites manifests directly in the form of distinct functional outcomes, including even opposite contribution of βarrs in signal-transduction for different receptors. Taken together, these findings uncover that despite an overall similar interaction and trafficking patterns; critical structural and functional differences exist in βarr complexes for different GPCRs that define and fine-tune receptor-specific downstream responses.

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Lactate as an Astroglial Signal Augmenting Aerobic Glycolysis and Lipid Metabolism

Frontiers in Physiology ◽

10.3389/fphys.2021.735532 ◽

2021 ◽

Vol 12 ◽

Author(s):

Anemari Horvat ◽

Robert Zorec ◽

Nina Vardjan

Keyword(s):

Lipid Metabolism ◽

Brain Function ◽

Lipid Droplets ◽

Aerobic Glycolysis ◽

Lactate Production ◽

G Protein Coupled Receptors ◽

Camp Production ◽

Signaling Mechanisms ◽

G Protein Coupled ◽

The Brain

Astrocytes, heterogeneous neuroglial cells, contribute to metabolic homeostasis in the brain by providing energy substrates to neurons. In contrast to predominantly oxidative neurons, astrocytes are considered primarily as glycolytic cells. They take up glucose from the circulation and in the process of aerobic glycolysis (despite the normal oxygen levels) produce L-lactate, which is then released into the extracellular space via lactate transporters and possibly channels. Astroglial L-lactate can enter neurons, where it is used as a metabolic substrate, or exit the brain via the circulation. Recently, L-lactate has also been considered to be a signaling molecule in the brain, but the mechanisms of L-lactate signaling and how it contributes to the brain function remain to be fully elucidated. Here, we provide an overview of L-lactate signaling mechanisms in the brain and present novel insights into the mechanisms of L-lactate signaling via G-protein coupled receptors (GPCRs) with the focus on astrocytes. We discuss how increased extracellular L-lactate upregulates cAMP production in astrocytes, most likely viaL-lactate-sensitive Gs-protein coupled GPCRs. This activates aerobic glycolysis, enhancing L-lactate production and accumulation of lipid droplets, suggesting that L-lactate augments its own production in astrocytes (i.e., metabolic excitability) to provide more L-lactate for neurons and that astrocytes in conditions of increased extracellular L-lactate switch to lipid metabolism.

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Receptor-activity-modifying protein 1 forms heterodimers with two G-protein-coupled receptors to define ligand recognition

Biochemical Journal ◽

10.1042/bj3510347 ◽

2000 ◽

Vol 351 (2) ◽

pp. 347-351 ◽

Cited By ~ 28

Author(s):

Kerstin LEUTHÄUSER ◽

Remo GUJER ◽

Amaya ALDECOA ◽

R. ANNE McKINNEY ◽

Roman MUFF ◽

...

Keyword(s):

Confocal Microscopy ◽

G Protein ◽

G Protein Coupled Receptors ◽

Receptor Activity ◽

Cross Linking ◽

Human Calcitonin ◽

Camp Production ◽

Surface Complexes ◽

Receptor Activity Modifying Proteins ◽

G Protein Coupled

Receptor-activity-modifying proteins (RAMPs) with single transmembrane domains define the function of two G-protein-coupled receptors of the B family. Cell-surface complexes of human RAMP1 (hRAMP1) and human calcitonin (CT) receptor isotype 2 (hCTR2) or rat CT-receptor-like receptor (rCRLR) have now been identified through protein cross-linking, co-immunoprecipitation and confocal microscopy. They are two distinct CT-gene-related peptide (CGRP) receptors coupled to cAMP production and pharmacologically distinguished by the CT and CGRP antagonists salmon CT(8-32) and human or rat CGRP(8-37). Thus direct molecular interactions of hRAMP1 with hCTR2 or rCRLR are required for CGRP recognition. hCTR2, moreover, adopts non-traditional functions through its association with hRAMP1.

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Allostery of atypical modulators at oligomeric G protein-coupled receptors

Scientific Reports ◽

10.1038/s41598-021-88399-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Rabindra V. Shivnaraine ◽

Brendan Kelly ◽

Gwendolynne Elmslie ◽

Xi-Ping Huang ◽

Yue John Dong ◽

...

Keyword(s):

G Protein ◽

Conformational Changes ◽

Radioligand Binding ◽

G Protein Coupled Receptors ◽

Oligomeric State ◽

Allosteric Sites ◽

Functional Consequences ◽

Orthosteric Ligands ◽

Dynamics Simulations ◽

G Protein Coupled

AbstractMany G protein-coupled receptors (GPCRs) are therapeutic targets, with most drugs acting at the orthosteric site. Some GPCRs also possess allosteric sites, which have become a focus of drug discovery. In the M2 muscarinic receptor, allosteric modulators regulate the binding and functional effects of orthosteric ligands through a mix of conformational changes, steric hindrance and electrostatic repulsion transmitted within and between the constituent protomers of an oligomer. Tacrine has been called an atypical modulator because it exhibits positive cooperativity, as revealed by Hill coefficients greater than 1 in its negative allosteric effect on binding and response. Radioligand binding and molecular dynamics simulations were used to probe the mechanism of that modulation in monomers and oligomers of wild-type and mutant M2 receptors. Tacrine is not atypical at monomers, which indicates that its atypical effects are a property of the receptor in its oligomeric state. These results illustrate that oligomerization of the M2 receptor has functional consequences.

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