scholarly journals Heterogeneity and dynamics of ERK and Akt activation by G protein-coupled receptors depend on the activated heterotrimeric G proteins

2021 ◽  
Author(s):  
Sergei Chavez-Abiega ◽  
Max Gronloh ◽  
Theodorus W.J. Gadella ◽  
Frank J. Bruggeman ◽  
Joachim Goedhart
Keyword(s):  
Single Cell ◽  
G Protein ◽  
G Proteins ◽  
Receptor Activation ◽  
Cellular Heterogeneity ◽  
Similar Response ◽  
Heterotrimeric G Proteins ◽  
Clustering Methods ◽  
Response Dynamics ◽  
Akt Activation

Kinases are fundamental regulators of cellular functions and play key roles in GPCR-mediated signaling pathways. Kinase activities are generally inferred from cell lysates, hiding the heterogeneity of the individual cellular responses to extracellular stimuli. Here, we study the dynamics and heterogeneity of ERK and Akt in genetically identical cells in response to activation of endogenously expressed GPCRs. We use kinase translocation reporters, high-content imaging, automated segmentation and clustering methods to assess cell-to-cell signaling heterogeneity. We observed ligand-concentration dependent response kinetics to histamine, α2-adrenergic, and S1P receptor stimulation that varied between cells. By using G protein inhibitors, we observed that Gq mediated the ERK and Akt responses to histamine. In contrast, Gi was necessary for ERK and Akt activation in response to α2-adrenergic receptor activation. ERK and Akt were also strongly activated by S1P, showing high heterogeneity at the single cell level, especially for ERK. In all cases, the cellular heterogeneity was not explained by distinct pre-stimulation levels or saturation of the measured response. Cluster analysis of time-series derived from 68,000 cells obtained under the different conditions revealed several distinct populations of cells that display similar response dynamics. The single-cell ERK responses to histamine and UK showed remarkably similar dynamics, despite the activation of different heterotrimeric G proteins. In contrast, the ERK response dynamics to S1P showed high heterogeneity, which was reduced by the inhibition of Gi. To conclude, we have set up an imaging and analysis strategy that reveals substantial cell-to-cell heterogeneity in kinase activity driven by GPCRs.

scholarly journals Every Detail Matters. That Is, How the Interaction between Gα Proteins and Membrane Affects Their Function

Membranes ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 222
Author(s):  
Agnieszka Polit ◽  
Paweł Mystek ◽  
Ewa Błasiak
Keyword(s):  
Signal Transduction ◽  
G Protein ◽  
G Proteins ◽  
Lipid Membranes ◽  
Signaling Proteins ◽  
Heterotrimeric G Proteins ◽  
Membrane Attachment ◽  
The Individual ◽  
Multicellular Organisms ◽  
G Protein Coupled

In highly organized multicellular organisms such as humans, the functions of an individual cell are dependent on signal transduction through G protein-coupled receptors (GPCRs) and subsequently heterotrimeric G proteins. As most of the elements belonging to the signal transduction system are bound to lipid membranes, researchers are showing increasing interest in studying the accompanying protein–lipid interactions, which have been demonstrated to not only provide the environment but also regulate proper and efficient signal transduction. The mode of interaction between the cell membrane and G proteins is well known. Despite this, the recognition mechanisms at the molecular level and how the individual G protein-membrane attachment signals are interrelated in the process of the complex control of membrane targeting of G proteins remain unelucidated. This review focuses on the mechanisms by which mammalian Gα subunits of G proteins interact with lipids and the factors responsible for the specificity of membrane association. We summarize recent data on how these signaling proteins are precisely targeted to a specific site in the membrane region by introducing well-defined modifications as well as through the presence of polybasic regions within these proteins and interactions with other components of the heterocomplex.

scholarly journals Signaling from G Protein-coupled Receptors to ERK5/Big MAPK 1 Involves Gαqand Gα12/13Families of Heterotrimeric G Proteins

2000 ◽  
Vol 275 (28) ◽  
pp. 21730-21736 ◽  
Author(s):  
Shigetomo Fukuhara ◽  
Maria Julia Marinissen ◽  
Mario Chiariello ◽  
J. Silvio Gutkind
Keyword(s):  
G Protein ◽  
G Proteins ◽  
G Protein Coupled Receptors ◽  
Heterotrimeric G Proteins ◽  
G Protein Coupled

scholarly journals Physiological Regulation of G Protein-Linked Signaling

1999 ◽  
Vol 79 (4) ◽  
pp. 1373-1430 ◽  
Author(s):  
Andrew J. Morris ◽  
Craig C. Malbon
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Chemokine Receptors ◽  
Transcriptional Control ◽  
Heterotrimeric G Proteins ◽  
Protein Prenylation ◽  
Physiological Control ◽  
Physiological Regulation ◽  
Surface Receptors ◽  
Genes Encoding

Heterotrimeric G proteins in vertebrates constitute a family molecular switches that transduce the activation of a populous group of cell-surface receptors to a group of diverse effector units. The receptors include the photopigments such as rhodopsin and prominent families such as the adrenergic, muscarinic acetylcholine, and chemokine receptors involved in regulating a broad spectrum of responses in humans. Signals from receptors are sensed by heterotrimeric G proteins and transduced to effectors such as adenylyl cyclases, phospholipases, and various ion channels. Physiological regulation of G protein-linked receptors allows for integration of signals that directly or indirectly effect the signaling from receptor→G protein→effector(s). Steroid hormones can regulate signaling via transcriptional control of the activities of the genes encoding members of G protein-linked pathways. Posttranscriptional mechanisms are under physiological control, altering the stability of preexisting mRNA and affording an additional level for regulation. Protein phosphorylation, protein prenylation, and proteolysis constitute major posttranslational mechanisms employed in the physiological regulation of G protein-linked signaling. Drawing upon mechanisms at all three levels, physiological regulation permits integration of demands placed on G protein-linked signaling.

scholarly journals Heterotrimeric G-Proteins Are Indispensable for FLT3-ITD autophosphorylation and Oncogenic Function

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2712-2712
Author(s):  
Maike Rehage ◽  
Susanne Wingert ◽  
Nadine Haetscher ◽  
Sabrina Bothur ◽  
Hubert Serve ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
G Protein ◽  
G Proteins ◽  
B Cell Lymphoma ◽  
Physical Interaction ◽  
Heterotrimeric G Proteins ◽  
Leukemic Transformation ◽  
Hematopoietic Stem ◽  
Oncogenic Function

Abstract Heterotrimeric G-proteins transmit signals of G-protein coupled receptors and regulate many basic cellular functions. However, their role in normal and malignant hematopoietic stem cells remains obscure. Activating mutations in the heterotrimeric G-protein Gaq were found in various cancers and its expression is enhanced in diffuse large B-cell lymphoma and T-ALL. Our previous data suggested the involvement of heterotrimeric G-proteins in Flt3-ITD-mediated leukemic transformation. FMS-like tyrosine kinase 3 with internal tandem duplication (FLT3-ITD) is a frequent oncoprotein in acute myeloid leukemia causing constitutive active STAT5 signaling. Here, we investigated a novel role of Gaq in Flt3-ITD-induced leukemic transformation. We could show that Gaq is indispensable for aberrant FLT3-ITD activation and oncogenic function as Gaq activity is necessary to maintain the autophosphorylation of FLT3-ITD. Gaq abrogation resulted in diminished cell proliferation and colony formation as well as delayed leukemogenesis in vivo of Flt3-ITD leukemic cells. Importantly, the growth inhibition could be rescued by addition of IL3 and did not occur in the presence of FLT3 ligand-activated FLT3 wildtype receptor, demonstrating the specificity of Gaq requirement for FLT3-ITD oncogenic signaling. Interestingly, co-immunoprecipitations revealed a direct physical interaction between FLT3-ITD and Gaq which did not require phosphorylation of the receptor tyrosine kinase. Hence, FLT3-ITD hyperphosphorylation seems to be rather a consequence of the interaction than a prerequisite. Flt3-ITD-induced transformation of murine hematopoietic stem/progenitor cells (HSPCs) strictly depended on the presence of Gaq, and the ablation of Gaq/11 in transplanted Flt3-ITD-transduced HSPCs from conditional Gaq/11 double knock-out mice delayed leukemic burden. These findings of an unexpected, yet critical, role of Gaq place the molecule as an important target for antileukemic strategies. Disclosures No relevant conflicts of interest to declare.

scholarly journals Minireview: GPCR and G Proteins: Drug Efficacy and Activation in Live Cells

2009 ◽  
Vol 23 (5) ◽  
pp. 590-599 ◽  
Author(s):  
Jean-Pierre Vilardaga ◽  
Moritz Bünemann ◽  
Timothy N. Feinstein ◽  
Nevin Lambert ◽  
Viacheslav O. Nikolaev ◽  
...  
Keyword(s):  
Energy Transfer ◽  
G Protein ◽  
G Proteins ◽  
Intracellular Signaling ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Activation ◽  
Live Cells ◽  
Mechanical Stimuli ◽  
G Protein Coupled

Abstract Many biochemical pathways are driven by G protein-coupled receptors, cell surface proteins that convert the binding of extracellular chemical, sensory, and mechanical stimuli into cellular signals. Their interaction with various ligands triggers receptor activation that typically couples to and activates heterotrimeric G proteins, which in turn control the propagation of secondary messenger molecules (e.g. cAMP) involved in critically important physiological processes (e.g. heart beat). Successful transfer of information from ligand binding events to intracellular signaling cascades involves a dynamic interplay between ligands, receptors, and G proteins. The development of Förster resonance energy transfer and bioluminescence resonance energy transfer-based methods has now permitted the kinetic analysis of initial steps involved in G protein-coupled receptor-mediated signaling in live cells and in systems as diverse as neurotransmitter and hormone signaling. The direct measurement of ligand efficacy at the level of the receptor by Förster resonance energy transfer is also now possible and allows intrinsic efficacies of clinical drugs to be linked with the effect of receptor polymorphisms.

Multiple mechanisms of receptor-G protein signaling specificity

1995 ◽  
Vol 269 (2) ◽  
pp. F141-F158 ◽  
Author(s):  
J. R. Raymond
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Linear Models ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
Heterotrimeric G Proteins ◽  
Cellular Functions ◽  
Signaling Specificity ◽  
Intracellular Signals ◽  
Specific Manner

The hormone-receptor-G protein complex transduces extracellular information into intracellular signals that ultimately regulate cellular functions in a highly specific manner. There are hundreds of receptor types that transduce signals through a relatively limited repertoire of heterotrimeric G proteins. Linear models of signaling specificity that require specific and highly selective coupling of hormone to receptor to G protein have proven inadequate to explain how highly particular signals are funneled through the G protein "bottleneck." Recent studies have uncovered a plethora of mechanisms that contribute to signaling specificity. This review focuses on the mechanisms that contribute to specificity in the interactions of receptors with G proteins.

Endothelin-B receptors activate Gα13

1999 ◽  
Vol 276 (4) ◽  
pp. C930-C937 ◽  
Author(s):  
Kenichiro Kitamura ◽  
Naoki Shiraishi ◽  
William D. Singer ◽  
Mary E. Handlogten ◽  
Kimio Tomita ◽  
...  
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Guanine Nucleotides ◽  
Heterotrimeric G Proteins ◽  
Human Embryonic Kidney ◽  
Cellular Effects ◽  
293 Cells ◽  
Specific Antagonist ◽  
Α Chain ◽  
Endothelin B

Endothelin (ET) receptors activate heterotrimeric G proteins that are members of the Gi, Gq, and Gs families but may also activate members of other families such as Gα12/13. Gα13 has multiple complex cellular effects that are similar to those of ET. We studied the ability of ET receptors to activate Gα13 using an assay for G protein α-chain activation that is based on the fact that an activated (GTP-bound) α-chain is resistant to trypsinization compared with an inactive (GDP-bound) α-chain. Nonhydrolyzable guanine nucleotides and AlMgF protected Gα13 from degradation by trypsin. In membranes from human embryonic kidney 293 cells that coexpress ETB receptors and α13, ET-3 and 5′-guanylylimidodiphosphate [Gpp(NH)p] increased the protection of α13 compared with Gpp(NH)p alone. The specificity of ETBreceptor-α13 coupling was documented by showing that β2receptors and isoproterenol or ETAreceptors and ET-1 did not activate α13 and that a specific antagonist for ETB receptors blocked ET-3-dependent activation of α13.

Single-cell RNA-seq data clustering: A survey with performance comparison study

2020 ◽  
Vol 18 (04) ◽  
pp. 2040005
Author(s):  
Ruiyi Li ◽  
Jihong Guan ◽  
Shuigeng Zhou
Keyword(s):  
Single Cell ◽  
Data Clustering ◽  
Performance Metrics ◽  
Clustering Algorithms ◽  
Cell Types ◽  
Performance Comparison ◽  
Cellular Heterogeneity ◽  
Clustering Methods ◽  
Multiple Perspectives ◽  
Underlying Mechanisms

Clustering analysis has been widely applied to single-cell RNA-sequencing (scRNA-seq) data to discover cell types and cell states. Algorithms developed in recent years have greatly helped the understanding of cellular heterogeneity and the underlying mechanisms of biological processes. However, these algorithms often use different techniques, were evaluated on different datasets and compared with some of their counterparts usually using different performance metrics. Consequently, there lacks an accurate and complete picture of their merits and demerits, which makes it difficult for users to select proper algorithms for analyzing their data. To fill this gap, we first do a review on the major existing scRNA-seq data clustering methods, and then conduct a comprehensive performance comparison among them from multiple perspectives. We consider 13 state of the art scRNA-seq data clustering algorithms, and collect 12 publicly available real scRNA-seq datasets from the existing works to evaluate and compare these algorithms. Our comparative study shows that the existing methods are very diverse in performance. Even the top-performance algorithms do not perform well on all datasets, especially those with complex structures. This suggests that further research is required to explore more stable, accurate, and efficient clustering algorithms for scRNA-seq data.

Ion Channels as G Protein Effectors

Physiology ◽  
1991 ◽  
Vol 6 (4) ◽  
pp. 158-161
Author(s):  
AM Brown
Keyword(s):  
Membrane Potential ◽  
Ion Channels ◽  
Cell Signaling ◽  
G Protein ◽  
G Proteins ◽  
Heterotrimeric G Proteins ◽  
Regulatory Processes ◽  
Functional Implications

Signaling between cells may be accomplished or accompanied by changes in membrane potential. The latter is regulated by ion channels, which are targets for regulatory processes initiated during signaling. Cell signaling frequently involves heterotrimeric G proteins. Evidence that ion channels are G protein effectors and functional implications of such regulation are reviewed.

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