scholarly journals Activation of Intracellular Signaling Pathways by the Murine Cytomegalovirus G Protein-Coupled Receptor M33 Occurs via PLC-β/PKC-Dependent and -Independent Mechanisms

2009 ◽  
Vol 83 (16) ◽  
pp. 8141-8152 ◽  
Author(s):  
Joseph D. Sherrill ◽  
Melissa P. Stropes ◽  
Olivia D. Schneider ◽  
Diana E. Koch ◽  
Fabiola M. Bittencourt ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Protein Kinase ◽  
Signaling Pathways ◽  
G Protein ◽  
Intracellular Signaling ◽  
Murine Cytomegalovirus ◽  
Dependent Manner ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled

ABSTRACT The presence of numerous G protein-coupled receptor (GPCR) homologs within the herpesvirus genomes suggests an essential role for these genes in viral replication in the infected host. Such is the case for murine cytomegalovirus (MCMV), where deletion of the M33 GPCR or replacement of M33 with a signaling defective mutant has been shown to severely attenuate replication in vivo. In the present study we utilized a genetically altered version of M33 (termed R131A) in combination with pharmacological inhibitors to further characterize the mechanisms by which M33 activates downstream signaling pathways. This R131A mutant of M33 fails to support salivary gland replication in vivo and, as such, is an important tool that can be used to examine the signaling activities of M33. We show that M33 stimulates the transcription factor CREB via heterotrimeric Gq/11 proteins and not through promiscuous coupling of M33 to the Gs pathway. Using inhibitors of signaling molecules downstream of Gq/11, we demonstrate that M33 stimulates CREB transcriptional activity in a phospholipase C-β and protein kinase C (PKC)-dependent manner. Finally, utilizing wild-type and R131A versions of M33, we show that M33-mediated activation of other signaling nodes, including the mitogen-activated protein kinase family member p38α and transcription factor NF-κB, occurs in the absence of Gq/11 and PKC signaling. The results from the present study indicate that M33 utilizes multiple mechanisms to modulate intracellular signaling cascades and suggest that signaling through PLC-β and PKC plays a central role in MCMV pathogenesis in vivo.

Glucocorticoid acts on a putative G protein-coupled receptor to rapidly regulate the activity of NMDA receptors in hippocampal neurons

2012 ◽  
Vol 302 (7) ◽  
pp. E747-E758 ◽  
Author(s):  
Yanmin Zhang ◽  
Hui Sheng ◽  
Jinshun Qi ◽  
Bei Ma ◽  
Jihu Sun ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nmda Receptors ◽  
G Protein ◽  
Hippocampal Neurons ◽  
Long Term Potentiation ◽  
G Protein Coupled Receptor ◽  
Protein Activity ◽  
Ca1 Region ◽  
G Protein Coupled

Glucocorticoids (GCs) have been demonstrated to act through both genomic and nongenomic mechanisms. The present study demonstrated that corticosterone rapidly suppressed the activity of N-methyl-d-aspartate (NMDA) receptors in cultured hippocampal neurons. The effect was maintained with corticosterone conjugated to bovine serum albumin and blocked by inhibition of G protein activity with intracellular GDP-β-S application. Corticosterone increased GTP-bound Gs protein and cyclic AMP (cAMP) production, activated phospholipase Cβ3 (PLC-β3), and induced inositol-1,4,5-triphosphate (IP3) production. Blocking PLC and the downstream cascades with PLC inhibitor, IP3 receptor antagonist, Ca2+ chelator, and protein kinase C (PKC) inhibitors prevented the actions of corticosterone. Blocking adenylate cyclase (AC) and protein kinase A (PKA) caused a decrease in NMDA-evoked currents. Application of corticosterone partly reversed the inhibition of NMDA currents caused by blockage of AC and PKA. Intracerebroventricular administration of corticosterone significantly suppressed long-term potentiation (LTP) in the CA1 region of the hippocampus within 30 min in vivo, implicating the possibly physiological significance of rapid effects of GC on NMDA receptors. Taken together, our results indicate that GCs act on a putative G protein-coupled receptor to activate multiple signaling pathways in hippocampal neurons, and the rapid suppression of NMDA activity by GCs is dependent on PLC and downstream signaling.

Multiple kinases phosphorylate the pancreatic cholecystokinin receptor in an agonist-dependent manner

1993 ◽  
Vol 264 (5) ◽  
pp. G840-G847 ◽  
Author(s):  
L. K. Gates ◽  
C. D. Ulrich ◽  
L. J. Miller
Keyword(s):  
Protein Kinase ◽  
G Protein ◽  
Pancreatic Acinar Cell ◽  
Dependent Manner ◽  
Receptor Kinase ◽  
G Protein Coupled Receptor ◽  
Guanine Nucleotide Binding Protein ◽  
Permeabilized Cells ◽  
High Concentrations ◽  
G Protein Coupled

The cholecystokinin (CCK) receptor on the rat pancreatic acinar cell is a guanine nucleotide-binding protein (G protein)-coupled receptor, which was recently demonstrated to be phosphorylated in response to agonist stimulation (Klueppelberg et al., J. Biol. Chem. 266: 17744-17746, 1991). In this work, we establish that this receptor is phosphorylated in response to a variety of homologous and heterologous secretagogues and that these phosphorylation events represent action by more than one protein kinase. One subgroup of kinases includes one or more isotype of protein kinase C (PKC), and is capable of playing a role in homologous and heterologous desensitization. A second subgroup of kinases that acts on the CCK receptor was defined by its resistance to 10 microM staurosporine, which was shown to inhibit all PKC in these cells. The activity of the second group of kinases was observed only in response to occupation of the CCK receptor by high concentrations of native hormone, raising the possibility of a "receptor-specific kinase." Similar to the prototypical kinase, beta-adrenergic receptor kinase (beta-ARK), this activity was inhibited in permeabilized cells by heparin. Furthermore, like this enzyme activity, beta-ARK was shown to be resistant to staurosporine. Based on its action on a G protein-coupled receptor, its activation at high concentrations of native agonist, and its pattern of inhibition, we believe that the staurosporine-insensitive CCK receptor kinase activity represents either beta-ARK or a closely related member of the receptor-specific kinase enzyme family.

scholarly journals Deletion of the R78 G Protein-Coupled Receptor Gene from Rat Cytomegalovirus Results in an Attenuated, Syncytium-Inducing Mutant Strain

1999 ◽  
Vol 73 (9) ◽  
pp. 7218-7230 ◽  
Author(s):  
Patrick S. Beisser ◽  
Gert Grauls ◽  
Cathrien A. Bruggeman ◽  
Cornelis Vink
Keyword(s):  
G Protein ◽  
Recombinant Virus ◽  
Murine Cytomegalovirus ◽  
Striking Difference ◽  
Predicted Amino Acid Sequence ◽  
G Protein Coupled Receptor ◽  
Recombinant Viruses ◽  
G Protein Coupled

ABSTRACT The rat cytomegalovirus (RCMV) R78 gene belongs to an uncharacterized class of viral G protein-coupled receptor (GCR) genes. The predicted amino acid sequence of the R78 open reading frame (ORF) shows 25 and 20% similarity with the gene products of murine cytomegalovirus M78 and human cytomegalovirus UL78, respectively. The R78 gene is transcribed throughout the early and late phases of infection in rat embryo fibroblasts (REF) in vitro. Transcription of R78 was found to result in three different mRNAs: (i) a 1.8-kb mRNA containing the R78 sequence, (ii) a 3.7-kb mRNA containing both R77 and R78 sequences, and (iii) a 5.7-kb mRNA containing at least ORF R77 and ORF R78 sequences. To investigate the function of the R78 gene, we generated two different recombinant virus strains: an RCMV R78 null mutant (RCMVΔR78a) and an RCMV mutant encoding a GCR from which the putative intracellular C terminus has been deleted (RCMVΔR78c). These recombinant viruses replicated with a 10- to 100-fold-lower efficiency than wild-type (wt) virus in vitro. Interestingly, unlike wt virus-infected REF, REF infected with the recombinants develop a syncytium-like appearance. A striking difference between wt and recombinant viruses was also seen in vivo: a considerably higher survival was seen among recombinant virus-infected rats than among RCMV-infected rats. We conclude that the RCMV R78 gene encodes a novel GCR-like polypeptide that plays an important role in both RCMV replication in vitro and the pathogenesis of viral infection in vivo.

Induction of osteoblast differentiation indexes by PTHrP in MG-63 cells involves multiple signaling pathways

2001 ◽  
Vol 281 (3) ◽  
pp. E489-E499 ◽  
Author(s):  
Luisa Carpio ◽  
Julienne Gladu ◽  
David Goltzman ◽  
Shafaat A. Rabbani
Keyword(s):  
Alkaline Phosphatase ◽  
Protein Kinase ◽  
Signaling Pathways ◽  
G Protein ◽  
Osteoblast Differentiation ◽  
Type I Collagen ◽  
Type I ◽  
Mutant Form ◽  
Dependent Manner ◽  
G Protein Coupled

Parathyroid hormone (PTH)-related peptide (PTHrP) can modulate the proliferation and differentiation of a number of cell types including osteoblasts. PTHrP can activate a G protein-coupled PTH/PTHrP receptor, which can interface with several second-messenger systems. In the current study, we have examined the signaling pathways involved in stimulated type I collagen and alkaline phosphatase expression in the human osteoblast-derived osteosarcoma cells, MG-63. By use of Northern blotting and histochemical analysis, maximum induction of these two markers of osteoblast differentiation occurred after 8 h of treatment with 100 nM PTHrP-(1–34). Chemical inhibitors of adenylate cyclase (H-89) or of protein kinase C (chelerythrine chloride) each diminished PTHrP-mediated type I collagen and alkaline phosphatase stimulation in a dose-dependent manner. These effects of PTHrP could also be blocked by inhibiting the Ras-mitogen-activated protein kinase (MAPK) pathway with a Ras farnesylation inhibitor, B1086, or with a MAPK inhibitor, PD-98059. Transient transfection of MG-63 cells with a mutant form of Gα, which can sequester βγ-subunits, showed significant downregulation of PTHrP-stimulated type I collagen expression, as did inhibition of phosphatidylinositol 3-kinase (PI 3-kinase) by wortmannin. Consequently, the βγ-PI 3-kinase pathway may be involved in PTHrP stimulation of Ras. Collectively, these results demonstrate that, acting via its G protein-coupled receptor, PTHrP can induce indexes of osteoblast differentiation by utilizing multiple, perhaps parallel, signaling pathways.

scholarly journals Kaposi's Sarcoma-Associated Herpesvirus-Encoded G Protein-Coupled Receptor ORF74 Constitutively Activates p44/p42 MAPK and Akt via Gi and Phospholipase C-Dependent Signaling Pathways

2002 ◽  
Vol 76 (4) ◽  
pp. 1744-1752 ◽  
Author(s):  
Martine J. Smit ◽  
Dennis Verzijl ◽  
Paola Casarosa ◽  
Marjon Navis ◽  
Henk Timmerman ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Signaling Pathways ◽  
Phospholipase C ◽  
G Protein ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
G Protein Coupled Receptor ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
G Protein Coupled

ABSTRACT The G protein-coupled receptor encoded by Kaposi's sarcoma-associated herpesvirus, also referred to as ORF74, has been shown to stimulate oncogenic and angiogenic signaling pathways in a constitutively active manner. The biochemical routes linking ORF74 to these signaling pathways are poorly defined. In this study, we show that ORF74 constitutively activates p44/p42 mitogen-activated protein kinase (MAPK) and Akt via Gi- and phospholipase C (PLC)-mediated signaling pathways. Activation of Akt by ORF74 appears to be phosphatidylinositol 3-kinase (PI3-K) dependent but, interestingly, is also mediated by activation of protein kinase C (PKC) and p44/p42 MAPK. ORF74 may signal to Akt via p44/p42 MAPK, which can be activated by Gi, through activation of PI3-K or through PKC via the PLC pathway. Signaling of ORF74 to these proliferative and antiapoptotic signaling pathways can be further modulated positively by growth-related oncogene (GROα/CXCL1) and negatively by human gamma interferon-inducible protein 10 (IP-10/CXCL10), thus acting as an agonist and an inverse agonist, respectively. Despite the ability of the cytomegalovirus-encoded chemokine receptor US28 to constitutively activate PLC, this receptor does not increase phosphorylation of p44/p42 MAPK or Akt in COS-7 cells. Hence, ORF74 appears to signal through a larger diversity of G proteins than US28, allowing it to couple to proliferative and antiapoptotic signaling pathways. ORF74 can therefore be envisioned as an attractive target for novel treatment of Kaposi's sarcoma.

scholarly journals Sulfotyrosines of the Kaposi's Sarcoma-Associated Herpesvirus G Protein-Coupled Receptor Promote Tumorigenesis through Autocrine Activation

2010 ◽  
Vol 84 (7) ◽  
pp. 3351-3361 ◽  
Author(s):  
Hao Feng ◽  
Zhifeng Sun ◽  
Michael R. Farzan ◽  
Pinghui Feng
Keyword(s):  
Signaling Pathways ◽  
G Protein ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Dependent Manner ◽  
G Protein Coupled Receptor ◽  
Akt Phosphorylation ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
G Protein Coupled

ABSTRACT The Kaposi's sarcoma-associated herpesvirus (KSHV) G protein-coupled receptor (vGPCR) is a bona fide signaling molecule that is implicated in KSHV-associated malignancies. Whereas vGPCR activates specific cellular signaling pathways in a chemokine-independent fashion, vGPCR binds a broad spectrum of CC and CXC chemokines, and the roles of chemokines in vGPCR tumorigenesis remain poorly understood. We report here that vGPCR is posttranslationally modified by sulfate groups at tyrosine residues within its N-terminal extracellular domain. A chemokine-binding assay demonstrated that the tyrosine sulfate moieties were critical for vGPCR association with GRO-α (an agonist) but not with IP-10 (an inverse agonist). A sulfated peptide corresponding to residues 12 through 33 of vGPCR, but not the unsulfated equivalent, partially inhibited vGPCR association with GRO-α. Although the vGPCR variant lacking sulfotyrosines activated downstream signaling pathways, the ability of the unsulfated vGPCR variant to induce tumor growth in nude mice was significantly diminished. Furthermore, the unsulfated vGPCR variant was unable to induce the secretion of proliferative cytokines, some of which serve as vGPCR agonists. This implies that autocrine activation by agonist chemokines is critical for vGPCR tumorigenesis. Indeed, GRO-α increased vGPCR-mediated AKT phosphorylation and vGPCR tumorigenesis in a sulfotyrosine-dependent manner. Our findings support the conclusion that autocrine activation triggered by chemokine agonists via sulfotyrosines is necessary for vGPCR tumorigenesis, thereby providing a rationale for future therapeutic design targeting the tumorigenic vGPCR.

Sign in / Sign up

Export Citation Format

Share Document