GRK2 promotes growth of medulloblastoma cells and protects them from chemotherapy-induced apoptosis

Abstract G-protein coupled receptor kinase 2 (GRK2; ADRBK1, BARK1) is most known as a regulator of G-protein coupled receptors. However, GRK2 also has other functions. Medulloblastomas are the most common malignant brain cancers in children. GRK2 has not been implicated in medulloblastoma biology. Here we report that GRK2 knockdown slowed cell growth, diminished proliferation, and enhanced cisplatin- and etoposide-induced apoptosis in medulloblastoma cell lines UW228-2 and Daoy. Reciprocally, GRK2 overexpression attenuated apoptosis induced by these chemotherapy drugs. Cisplatin and etoposide increased phosphorylation of AKT (S473) and GRK2 knockdown mitigated this increase. Cisplatin and etoposide attenuated ERK phosphorylation, but GRK2 knockdown did not alter this effect. Wildtype GRK2 reversed the increase in cisplatin- and etoposide-induced apoptosis caused by GRK2 knockdown. GRK2-K220R (kinase dead) and GRK2-S670A (unphosphorylated, constitutively active) conferred protection from cisplatin that was similar to wildtype GRK2, suggesting that this protection may be mediated though a kinase-independent activity of GRK2. These data demonstrate that GRK2 contributes to proliferation and survival of these medulloblastoma cell lines and to their protection from cisplatin- and etoposide-induced apoptosis.

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Faculty Opinions recommendation of Expression profiling of G-protein-coupled receptors in human urothelium and related cell lines.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.717648068.793455010 ◽

2012 ◽

Author(s):

David Grundy ◽

Marina Liaskos

Keyword(s):

G Protein ◽

Cell Lines ◽

Expression Profiling ◽

G Protein Coupled Receptors ◽

Related Cell ◽

G Protein Coupled

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GRKs as Modulators of Neurotransmitter Receptors

Cells ◽

10.3390/cells10010052 ◽

2020 ◽

Vol 10 (1) ◽

pp. 52

Author(s):

Eugenia V. Gurevich ◽

Vsevolod V. Gurevich

Keyword(s):

G Protein ◽

General Model ◽

G Protein Coupled Receptors ◽

Receptor Internalization ◽

Neurotransmitter Receptors ◽

Receptor Subtype ◽

Receptor Subtypes ◽

Receptor Kinase ◽

G Protein Coupled ◽

Homologous Desensitization

Many receptors for neurotransmitters, such as dopamine, norepinephrine, acetylcholine, and neuropeptides, belong to the superfamily of G protein-coupled receptors (GPCRs). A general model posits that GPCRs undergo two-step homologous desensitization: the active receptor is phosphorylated by kinases of the G protein-coupled receptor kinase (GRK) family, whereupon arrestin proteins specifically bind active phosphorylated receptors, shutting down G protein-mediated signaling, facilitating receptor internalization, and initiating distinct signaling pathways via arrestin-based scaffolding. Here, we review the mechanisms of GRK-dependent regulation of neurotransmitter receptors, focusing on the diverse modes of GRK-mediated phosphorylation of receptor subtypes. The immediate signaling consequences of GRK-mediated receptor phosphorylation, such as arrestin recruitment, desensitization, and internalization/resensitization, are equally diverse, depending not only on the receptor subtype but also on phosphorylation by GRKs of select receptor residues. We discuss the signaling outcome as well as the biological and behavioral consequences of the GRK-dependent phosphorylation of neurotransmitter receptors where known.

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A functional assay for G-protein-coupled receptors using stably transformed insect tissue culture cell lines

Analytical Biochemistry ◽

10.1016/s0003-2697(03)00354-3 ◽

2003 ◽

Vol 320 (1) ◽

pp. 88-103 ◽

Cited By ~ 32

Author(s):

Peter J.K Knight ◽

Tom A Pfeifer ◽

Tom A Grigliatti

Keyword(s):

Tissue Culture ◽

G Protein ◽

Cell Lines ◽

Culture Cell ◽

G Protein Coupled Receptors ◽

Tissue Culture Cell ◽

Functional Assay ◽

Insect Tissue Culture ◽

G Protein Coupled

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Specific oxylipins enhance vertebrate hematopoiesis via the receptor GPR132

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1806077115 ◽

2018 ◽

Vol 115 (37) ◽

pp. 9252-9257 ◽

Cited By ~ 17

Author(s):

Jamie L. Lahvic ◽

Michelle Ammerman ◽

Pulin Li ◽

Megan C. Blair ◽

Emma R. Stillman ◽

...

Keyword(s):

Fatty Acids ◽

G Protein ◽

Cell Lines ◽

Saturated Fatty Acids ◽

Receptor Activation ◽

G Protein Coupled Receptors ◽

Hydroxy Fatty Acids ◽

High Affinity ◽

G Protein Coupled

Epoxyeicosatrienoic acids (EETs) are lipid-derived signaling molecules with cardioprotective and vasodilatory actions. We recently showed that 11,12-EET enhances hematopoietic induction and engraftment in mice and zebrafish. EETs are known to signal via G protein-coupled receptors, with evidence supporting the existence of a specific high-affinity receptor. Identification of a hematopoietic-specific EET receptor would enable genetic interrogation of EET signaling pathways, and perhaps clinical use of this molecule. We developed a bioinformatic approach to identify an EET receptor based on the expression of G protein-coupled receptors in cell lines with differential responses to EETs. We found 10 candidate EET receptors that are expressed in three EET-responsive cell lines, but not expressed in an EET-unresponsive line. Of these, only recombinant GPR132 showed EET-responsiveness in vitro, using a luminescence-based β-arrestin recruitment assay. Knockdown of zebrafish gpr132b prevented EET-induced hematopoiesis, and marrow from GPR132 knockout mice showed decreased long-term engraftment capability. In contrast to high-affinity EET receptors, GPR132 is reported to respond to additional hydroxy-fatty acids in vitro, and we found that these same hydroxy-fatty acids enhance hematopoiesis in the zebrafish. We conducted structure–activity relationship analyses using both cell culture and zebrafish assays on diverse medium-chain fatty acids. Certain oxygenated, unsaturated free fatty acids showed high activation of GPR132, whereas unoxygenated or saturated fatty acids had lower activity. Absence of the carbon-1 position carboxylic acid prevented activity, suggesting that this moiety is required for receptor activation. GPR132 responds to a select panel of oxygenated polyunsaturated fatty acids to enhance both embryonic and adult hematopoiesis.

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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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