Lysophospholipid Growth Factors and Their G Protein-Coupled Receptors in Immunity, Coronary Artery Disease, and Cancer

The physiological lysophospholipids (LPLs), exemplified by lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), are omnific mediators of normal cellular proliferation, survival, and functions. Although both LPA and S1P attain micromolar concentrations in many biological fluids, numerous aspects of their biosynthesis, transport, and metabolic degradation are unknown. Eight members of a new subfamily of G protein-coupled LPA/S1P receptors, originally termed Edg Rs, bind either LPA or S1P with high affinity and transduce a series of growth-related and/or cytoskeleton-based functional responses. The most critical areas of LPL biology and pathobiology are neural development and neurodegeneration, immunity, atherosclerosis and myocardial injury, and cancer. Data from analyses of T cells established two basic points: (1) the plasticity and adaptability of expression of LPA/S1P Rs by some cells as a function of activation, and (2) the role of opposing signals from two different receptors for the same ligand as a mechanism for fine control of effects of LPLs. In the heart, LPLs may promote coronary atherosclerosis, but are effectively cytoprotective for hypoxic cardiac myocytes and those exposed to oxygen free radicals. The findings of production of LPA by some types of tumor cells, overexpression of selected sets of LPA receptors by the same tumor cells, and augmentation of the effects of protein growth factors by LPA have suggested pathogenetic roles for the LPLs in cancer. The breadth of physiologic and pathologic activities of LPLs emphasizes the importance of developing bioavailable nonlipid agonists and antagonists of the LPA/S1P receptors for diverse therapeutic applications.

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Development of a Novel SNAP-Epitope Tag/Near-Infrared Imaging Assay to Quantify G Protein-Coupled Receptor Degradation in Human Cells

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555220979793 ◽

2021 ◽

pp. 247255522097979

Author(s):

Kyung-Soon Lee ◽

Edelmar Navaluna ◽

Nicole M. Marsh ◽

Eric M. Janezic ◽

Chris Hague

Keyword(s):

G Protein ◽

Adrenergic Receptor ◽

Near Infrared ◽

Human Cells ◽

Receptor Subtypes ◽

G Protein Coupled Receptor ◽

Functional Responses ◽

Epitope Tag ◽

Nir Imaging ◽

G Protein Coupled

We have developed a novel reporter assay that leverages SNAP-epitope tag/near-infrared (NIR) imaging technology to monitor G protein-coupled receptor (GPCR) degradation in human cell lines. N-terminal SNAP-tagged GPCRs were subcloned and expressed in human embryonic kidney (HEK) 293 cells and then subjected to 24 h of cycloheximide (CHX)-chase degradation assays to quantify receptor degradation half-lives ( t1/2) using LICOR NIR imaging–polyacrylamide gel electrophoresis (PAGE) analysis. Thus far, we have used this method to quantify t1/2 for all nine adrenergic (ADRA1A, ADRA1B, ADRA1D, ADRA2A, ADRA2B, ADRA2C, ADRB1, ADRB2, ADRB3), five somatostatin (SSTR1, SSTR2, SSTR3, SSTR4, SSTR5), four chemokine (CXCR1, CXCR2, CXCR3, CXCR5), and three 5-HT2 (5HT2A, 5HT2B, 5HT2C) receptor subtypes. SNAP-GPCR-CHX degradation t1/2 values ranged from 0.52 h (ADRA1D) to 5.5 h (SSTR3). On the contrary, both the SNAP-tag alone and SNAP-tagged and endogenous β-actin were resistant to degradation with CHX treatment. Treatment with the proteasome inhibitor bortezomib produced significant but variable increases in SNAP-GPCR protein expression levels, indicating that SNAP-GPCR degradation primarily occurs through the proteasome. Remarkably, endogenous β2-adrenergic receptor/ADRB2 dynamic mass redistribution functional responses to norepinephrine were significantly decreased following CHX treatment, with a time course equivalent to that observed with the SNAP-ADRB2 degradation assay. We subsequently adapted this assay into a 96-well glass-bottom plate format to facilitate high-throughput GPCR degradation screening. t1/2 values quantified for the α1-adrenergic receptor subtypes (ADRA1A, ADRA1B, ADR1D) using the 96-well-plate format correlated with t1/2 values quantified using NIR-PAGE imaging analysis. In summary, this novel assay permits precise quantitative analysis of GPCR degradation in human cells and can be readily adapted to quantify degradation for any membrane protein of interest.

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Rgs4 is a regulator of mTOR activity required for motoneuron axon outgrowth and neuronal development in zebrafish

Scientific Reports ◽

10.1038/s41598-021-92758-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Aya Mikdache ◽

Marie-José Boueid ◽

Lorijn van der Spek ◽

Emilie Lesport ◽

Brigitte Delespierre ◽

...

Keyword(s):

Nervous System ◽

G Protein ◽

Neural Development ◽

Neuronal Development ◽

Axon Outgrowth ◽

Rgs Proteins ◽

Protein Signaling ◽

Loss Of Function ◽

G Protein Coupled

AbstractThe Regulator of G protein signaling 4 (Rgs4) is a member of the RGS proteins superfamily that modulates the activity of G-protein coupled receptors. It is mainly expressed in the nervous system and is linked to several neuronal signaling pathways; however, its role in neural development in vivo remains inconclusive. Here, we generated and characterized a rgs4 loss of function model (MZrgs4) in zebrafish. MZrgs4 embryos showed motility defects and presented reduced head and eye sizes, reflecting defective motoneurons axon outgrowth and a significant decrease in the number of neurons in the central and peripheral nervous system. Forcing the expression of Rgs4 specifically within motoneurons rescued their early defective outgrowth in MZrgs4 embryos, indicating an autonomous role for Rgs4 in motoneurons. We also analyzed the role of Akt, Erk and mechanistic target of rapamycin (mTOR) signaling cascades and showed a requirement for these pathways in motoneurons axon outgrowth and neuronal development. Drawing on pharmacological and rescue experiments in MZrgs4, we provide evidence that Rgs4 facilitates signaling mediated by Akt, Erk and mTOR in order to drive axon outgrowth in motoneurons and regulate neuronal numbers.

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Sustained muscle contraction induced by agonists, growth factors, and Ca2+ mediated by distinct PKC isozymes

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.2000.279.1.g201 ◽

2000 ◽

Vol 279 (1) ◽

pp. G201-G210 ◽

Cited By ~ 40

Author(s):

K. S. Murthy ◽

J. R. Grider ◽

J. F. Kuemmerle ◽

G. M. Makhlouf

Keyword(s):

Growth Factors ◽

G Protein ◽

Muscle Cells ◽

Sustained Contraction ◽

Calphostin C ◽

Epidermal Growth ◽

Pkc Isozymes ◽

G Protein Coupled ◽

Pkc Activation

The role of protein kinase C (PKC) in sustained contraction was examined in intestinal circular and longitudinal muscle cells. Initial contraction induced by agonists (CCK-8 and neuromedin C) was abolished by 1) inhibitors of Ca2+ mobilization (neomycin and dimethyleicosadienoic acid), 2) calmidazolium, and 3) myosin light chain (MLC) kinase (MLCK) inhibitor KT-5926. In contrast, sustained contraction was not affected by these inhibitors but was abolished by 1) the PKC inhibitors chelerythrine and calphostin C, 2) PKC-ε antibody, and 3) a pseudosubstrate PKC-ε inhibitor. GDPβS abolished both initial and sustained contraction, whereas a Gαq/11 antibody inhibited only initial contraction, implying that sustained contraction was dependent on activation of a distinct G protein. Sustained contraction induced by epidermal growth factor was inhibited by calphostin C, PKC-α,β,γ antibody, and a pseudosubstrate PKC-α inhibitor. Ca2+ (0.4 μM) induced an initial contraction in permeabilized muscle cells that was blocked by calmodulin and MLCK inhibitors and a sustained contraction that was blocked by calphostin C and a PKC-α,β,γ antibody. Thus initial contraction induced by Ca2+, agonists, and growth factors is mediated by MLCK, whereas sustained contraction is mediated by specific Ca2+-dependent and -independent PKC isozymes. G protein-coupled receptors are linked to PKC activation via distinct G proteins.

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Modulating Sphingosine-1-Phosphate receptors to improve chemotherapy delivery to Ewing sarcoma

10.1101/805655 ◽

2019 ◽

Author(s):

Enrica Marmonti ◽

Hannah Savage ◽

Aiqian Zhang ◽

Claudia Alvarez ◽

Miriam Morrell ◽

...

Keyword(s):

G Protein ◽

Ewing Sarcoma ◽

Tyrosine Kinases ◽

Tumor Vasculature ◽

Cell Junction ◽

Tumor Vessels ◽

S1p Receptors ◽

Vessel Permeability ◽

Sphingosine 1 Phosphate ◽

G Protein Coupled

ABSTRACTTumor vasculature is innately dysfunctional. Poorly functional tumor vessels inefficiently deliver chemotherapy to tumor cells; vessel hyper-permeability promotes chemotherapy delivery primarily to a tumor’s periphery. Here we identify a method for enhancing chemotherapy delivery and efficacy in Ewing sarcoma (ES) in mice by modulating tumor vessel permeability. Vessel permeability is partially controlled by the G protein-coupled Sphinosine-1-phosphate receptors 1 and 2 (S1PR1 and S1PR2) on endothelial cells. S1PR1 promotes endothelial cell junction integrity while S1PR2 destabilizes it. We hypothesize that an imbalance of S1PR1:S1PR2 is partially responsible for the dysfunctional vascular phenotype characteristic of ES and that by altering the balance in favor of S1PR1, ES vessel hyper-permeability can be reversed. In this study, we demonstrate that pharmacologic activation of S1PR1 by SEW2871 or inhibition of S1PR2 by JTE-013 caused more organized, mature, and functional tumor vessels. Importantly, S1PR1 activation or S1PR2 inhibition improved chemotherapy delivery to the tumor and anti-tumor efficacy. Our data suggests that pharmacologic targeting of S1PR1 and S1PR2 may be a useful adjuvant to standard chemotherapy for ES patients.NOVELTY AND IMPACTThis study demonstrates that Sphingosine-1-Phosphate (S1P) receptors are potential novel targets for tumor vasculature remodeling and adjuvant therapy for the treatment of Ewing Sarcoma. Unlike receptor tyrosine kinases that have already been extensively evaluated for use as vascular normalizing agents in oncology, S1P receptors are G protein-coupled receptors, which have not been well studied in tumor endothelium. Pharmacologic activators and inhibitors of S1P receptors are currently in clinical trials for treatment of auto-immune and cardiovascular diseases, indicating potential for clinical translation of this work.

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Distinct phosphorylation sites in a prototypical GPCR differently orchestrate β-arrestin interaction, trafficking, and signaling

Science Advances ◽

10.1126/sciadv.abb8368 ◽

2020 ◽

Vol 6 (37) ◽

pp. eabb8368 ◽

Cited By ~ 1

Author(s):

Hemlata Dwivedi-Agnihotri ◽

Madhu Chaturvedi ◽

Mithu Baidya ◽

Tomasz Maciej Stepniewski ◽

Shubhi Pandey ◽

...

Keyword(s):

G Protein ◽

Phosphorylation Site ◽

Dynamics Simulation ◽

Phosphorylation Sites ◽

Functional Responses ◽

Biased Signaling ◽

The Stability ◽

G Protein Coupled ◽

Structural Aspects

Agonist-induced phosphorylation of G protein–coupled receptors (GPCRs) is a key determinant for their interaction with β-arrestins (βarrs) and subsequent functional responses. Therefore, it is important to decipher the contribution and interplay of different receptor phosphorylation sites in governing βarr interaction and functional outcomes. Here, we find that several phosphorylation sites in the human vasopressin receptor (V2R), positioned either individually or in clusters, differentially contribute to βarr recruitment, trafficking, and ERK1/2 activation. Even a single phosphorylation site in V2R, suitably positioned to cross-talk with a key residue in βarrs, has a decisive contribution in βarr recruitment, and its mutation results in strong G-protein bias. Molecular dynamics simulation provides mechanistic insights into the pivotal role of this key phosphorylation site in governing the stability of βarr interaction and regulating the interdomain rotation in βarrs. Our findings uncover important structural aspects to better understand the framework of GPCR-βarr interaction and biased signaling.

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Sphingosine 1-Phosphate (S1P) Induces Migration and ERK/MAP-Kinase-Dependent Proliferation in Chronic Lymphocytic Leukemia (B-CLL) Due to Expression of the G Protein-Coupled Receptors S1P1/4.

Blood ◽

10.1182/blood.v106.11.4996.4996 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4996-4996

Author(s):

Gabriele Seitz ◽

Sedat Yildirim ◽

Andreas M. Boehmler ◽

Lothar Kanz ◽

Robert Möhle

Keyword(s):

Chronic Lymphocytic Leukemia ◽

G Protein ◽

Cell Lines ◽

Peripheral Blood ◽

G Protein Coupled Receptors ◽

Lymphocytic Leukemia ◽

S1p Receptors ◽

Sphingosine 1 Phosphate ◽

G Protein Coupled

Abstract Egress of lymphocytes from lymphoid organs into the circulation has been shown to depend on the presence of the lipid mediator sphingosine 1-phosphate (S1P) in the peripheral blood, and expression of corresponding S1P receptors (i.e., S1P1), that belong to the family of 7-transmembrane G protein-coupled receptors (GPCR). As circulating lymphocytic lymphoma cells are a hallmark of chronic lymphocytic leukemia, we analyzed expression of different S1P receptors and the effects of S1P on B-CLL cells. By qualitative and quantitative (TaqMan) RT-PCR, significant mRNA expression of S1P1 and S1P4 was found in CLL cell lines (EHEB, MEC-1) and in most samples (S1P1 in 88%, S1P4 in 100%) of primary CD19+ cells isolated from the peripheral blood of untreated B-CLL patients. mRNA of other S1P receptors (S1P2, S1P3, S1P5) was less consistently detected. Normal, nonmalignant B cells were strongly positive for S1P1, while other S1P receptors were weakly expressed or negative. S1P induced typical effects of chemotactic GPCR, such as actin polymerization (analyzed by flow cytometry) and chemotaxis (measured in a modified Boyden chamber assay) in CLL cell lines and primary B-CLL cells. After serum deprivation in vitro, S1P induced phosphorylation of ERK/MAP-kinase as analyzed by Western blot, demonstrating that S1P receptors expressed in CLL were able to activate signaling pathways of GPCR not only related to cell migration and chemotaxis, but also to cell proliferation. Of note, the S1P1 ligand FTY720, which induces receptor internalization after prolonged exposure and acts as an antagonist, resulted in apoptosis in CLL cell lines and primary CLL cells in vitro, as measured by MTT-test and staining with Annexin-FITC, respectively. We conclude that sphingosine 1-phosphate, which is present in the peripheral blood in considerable amounts, contributes to the trafficking of B-CLL cells expressing the GPCRs S1P1/4, and to their prolonged survival.

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Binding of α-melanocyte-stimulating hormone to its G-protein-coupled receptor on B-lymphocytes activates the Jak/STAT pathway

Biochemical Journal ◽

10.1042/bj3310211 ◽

1998 ◽

Vol 331 (1) ◽

pp. 211-216 ◽

Cited By ~ 82

Author(s):

Joseph J. BUGGY

Keyword(s):

G Protein ◽

Protein Tyrosine Kinase ◽

Cellular Proliferation ◽

B Lymphocyte ◽

Janus Kinase ◽

Lymphocyte Function ◽

Amino Acid Peptide ◽

Melanocyte Stimulating Hormone ◽

G Protein Coupled ◽

Stimulating Hormone

α-Melanocyte-stimulating hormone (α-MSH) is a 13-amino-acid peptide with a variety of physiological effects, including the stimulation of melanocyte proliferation and melanogenesis, temperature control, control of prolactin release and the modulation of cytokine action in the immune system. There are five known subtypes of G-protein-coupled receptors, which bind with different affinities to α-MSH. This paper provides evidence that Ba/F3 pro-B-lymphocyte cells express the gene for the melanocortin 5 (MC5) receptor and specifically bind α-MSH. Western-blot analysis reveals that α-MSH binding stimulates Janus kinase 2 (JAK2) and signal transducers and activators of transcription (STAT1) tyrosine phosphorylation in both Ba/F3 cells and human cultured IM-9 lymphocytes. α-MSH is further revealed to activate JAK2 in mouse L-cells stably expressing the human MC5 receptor. Finally, α-MSH binding is shown to result in an enhancement of cellular proliferation. These findings identify a new protein tyrosine kinase pathway in the action of α-MSH, and suggest that α-MSH plays an important role in B-lymphocyte function via the activation of the same intracellular phosphorylation pathway used by cytokines and growth factors.

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Ligand-specific conformational change of the G-protein-coupled receptor ALX/FPR2 determines proresolving functional responses

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1308253110 ◽

2013 ◽

Vol 110 (45) ◽

pp. 18232-18237 ◽

Cited By ~ 157

Author(s):

S. N. Cooray ◽

T. Gobbetti ◽

T. Montero-Melendez ◽

S. McArthur ◽

D. Thompson ◽

...

Keyword(s):

Conformational Change ◽

G Protein ◽

G Protein Coupled Receptor ◽

Functional Responses ◽

G Protein Coupled

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Smoothened Signal Transduction Is Promoted by G Protein-Coupled Receptor Kinase 2

Molecular and Cellular Biology ◽

10.1128/mcb.00546-06 ◽

2006 ◽

Vol 26 (20) ◽

pp. 7550-7560 ◽

Cited By ~ 91

Author(s):

Alison R. Meloni ◽

Gregory B. Fralish ◽

Patrick Kelly ◽

Ali Salahpour ◽

James K. Chen ◽

...

Keyword(s):

Signal Transduction ◽

G Protein ◽

Sonic Hedgehog ◽

Cellular Proliferation ◽

Receptor Kinase ◽

G Protein Coupled Receptor ◽

Positive Role ◽

Proliferation And Differentiation ◽

Hedgehog Proteins ◽

G Protein Coupled

ABSTRACT Deregulation of the Sonic hedgehog pathway has been implicated in an increasing number of human cancers. In this pathway, the seven-transmembrane (7TM) signaling protein Smoothened regulates cellular proliferation and differentiation through activation of the transcription factor Gli. The activity of mammalian Smoothened is controlled by three different hedgehog proteins, Indian, Desert, and Sonic hedgehog, through their interaction with the Smoothened inhibitor Patched. However, the mechanisms of signal transduction from Smoothened are poorly understood. We show that a kinase which regulates signaling by many “conventional” 7TM G-protein-coupled receptors, G protein-coupled receptor kinase 2 (GRK2), participates in Smoothened signaling. Expression of GRK2, but not catalytically inactive GRK2, synergizes with active Smoothened to mediate Gli-dependent transcription. Moreover, knockdown of endogenous GRK2 by short hairpin RNA (shRNA) significantly reduces signaling in response to the Smoothened agonist SAG and also inhibits signaling induced by an oncogenic Smoothened mutant, Smo M2. We find that GRK2 promotes the association between active Smoothened and β-arrestin 2. Indeed, Gli-dependent signaling, mediated by coexpression of Smoothened and GRK2, is diminished by β-arrestin 2 knockdown with shRNA. Together, these data suggest that GRK2 plays a positive role in Smoothened signaling, at least in part, through the promotion of an association between β-arrestin 2 and Smoothened.

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Nuclear G protein-coupled oestrogen receptor (GPR30) predicts poor survival in patients with ovarian cancer

Journal of International Medical Research ◽

10.1177/0300060517717625 ◽

2017 ◽

Vol 46 (2) ◽

pp. 723-731 ◽

Cited By ~ 10

Author(s):

Cai-xia Zhu ◽

Wei Xiong ◽

Ma-lie Wang ◽

Juan Yang ◽

Hui-juan Shi ◽

...

Keyword(s):

Ovarian Cancer ◽

Overall Survival ◽

G Protein ◽

Oestrogen Receptor ◽

Advanced Ovarian Cancer ◽

High Grade ◽

Tissue Samples ◽

Intracellular Location ◽

Cancer Data ◽

G Protein Coupled

Objective To demonstrate the correlation between nuclear and cytoplasmic G protein-coupled oestrogen receptor (GPR30) expression and clinicopathological features and outcome in patients with ovarian cancer. Methods Nuclear and cytoplasmic GPR30 expressions were determined using immunohistochemistry to identify the intracellular location in tissues from patients with ovarian cancer. Data were correlated with clinicopathological characteristics and outcomes. Results Tissue samples were obtained from 110 patients with epithelial ovarian cancer between 2005 and 2010. Nuclear GPR30 was significantly more frequent in the group of patients with recurrence. The presence of nuclear GPR30 predicted lower overall survival) and 5-year progression-free survival in all patients with ovarian cancer and overall survival in patients with high grade ovarian cancer. Cytoplasmic GPR30 was observed significantly more often in advanced ovarian cancer and did not predict survival. Conclusion This study showed that nuclear GPR30 is an independent negative prognostic indicator in patients with ovarian cancer, especially in those with a high grade malignancy.

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