A Dominant Negative Mutant of the G Protein-Coupled Receptor Kinase 2 Selectively Attenuates Adenosine A2Receptor Desensitization

1997 ◽  
Vol 51 (6) ◽  
pp. 991-998 ◽  
Author(s):  
Stuart J. Mundell ◽  
Jeffrey L. Benovic ◽  
Eamonn Kelly
Keyword(s):  
G Protein ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Receptor Kinase ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled ◽  
Negative Mutant

scholarly journals Role of G Protein-Coupled Receptor Kinases on the Agonist-Induced Phosphorylation and Internalization of the Follitropin Receptor

1999 ◽  
Vol 13 (6) ◽  
pp. 866-878 ◽  
Author(s):  
Maria de Fatima M. Lazari ◽  
Xuebo Liu ◽  
Kazuto Nakamura ◽  
Jeffrey L. Benovic ◽  
Mario Ascoli
Keyword(s):  
Cell Line ◽  
G Protein ◽  
Dominant Negative ◽  
Receptor Internalization ◽  
Dominant Negative Mutant ◽  
Deficient Mutant ◽  
G Protein Coupled Receptor ◽  
Western Blots ◽  
293 Cells ◽  
G Protein Coupled

Abstract The experiments presented herein were designed to identify members of the G protein-coupled receptor kinase (GRK) family that participate in the agonist-induced phosphorylation and internalization of the rat FSH receptor (rFSHR). Western blots of human kidney 293 cells (the cell line used in transfection experiments) and MSC-1 cells (a cell line derived from Sertoli cells that displays many of the differentiated functions of their normal counterparts) reveal the presence of GRK2 and GRK6 in both cell lines as well as GRK4 in MSC-1 cells. Cotransfection of 293 cells with the rFSHR and GRK2, GRK4α, or GRK6 resulted in an increase in the agonist-induced phosphorylation of the rFSHR. Cotransfections of the rFSHR with GRKs or arrestin-3 enhanced the agonist-induced internalization of the rFHSR, and combinations of GRKs and arrestin-3 were more effective than the individual components. To characterize the involvement of endogenous GRKs on phosphorylation and internalization, we inhibited endogenous GRK2 by overexpression of a kinase-deficient mutant of GRK2 or Gαt, a scavenger of Gβγ. We also inhibited endogenous GRK6 by overexpression of a kinase-deficient mutant of GKR6. All three constructs were effective inhibitors of phosphorylation, but only the kinase-deficient mutant of GRK2 and Gαt inhibited internalization. The inhibition of internalization induced by these two constructs was less pronounced than that induced by a dominant-negative mutant of the nonvisual arrrestins, however. The finding that inhibitors of GRK2 and GRK6 impair phosphorylation, but only the inhibitors of GRK2 impair internalization, suggests that different GRKs have differential effects on receptor internalization.

scholarly journals Fractalkine stimulates angiogenesis by activating the Raf-1/MEK/ERK- and PI3K/Akt/eNOS-dependent signal pathways

2006 ◽  
Vol 291 (6) ◽  
pp. H2836-H2846 ◽  
Author(s):  
Seon-Jin Lee ◽  
Seung Namkoong ◽  
Young-Mi Kim ◽  
Chun-Ki Kim ◽  
Hansoo Lee ◽  
...  
Keyword(s):  
G Protein ◽  
Umbilical Vein ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Signal Pathways ◽  
No Production ◽  
G Protein Coupled Receptor ◽  
Angiogenic Activity ◽  
Enos Phosphorylation ◽  
G Protein Coupled

Fractalkine (FKN) has been implicated in modulation of angiogenesis and vascular inflammation, but the underlying mechanism has not been elucidated. We have investigated the molecular mechanism by which FKN regulates angiogenesis. We found that recombinant FKN increases in vitro proliferation, migration, and tube formation of human umbilical vein endothelial cells and stimulates in vivo angiogenesis. FKN-induced angiogenesis was accompanied by phosphorylation of ERK, Akt, and endothelial nitric oxide (NO) synthase (eNOS), as well as an increase in NO production. These biochemical events and angiogenesis were completely inhibited by the G protein-coupled receptor inhibitor pertussis toxin. Inhibitors of Raf-1, MEK, phosphatidylinositol 3-kinase (PI3K), and eNOS or transfection with dominant-negative forms of ERK and Akt significantly suppressed the angiogenic activity of FKN. However, inhibitors of Raf-1 and MEK or a dominant-negative ERK mutant blocked FKN-induced ERK, but not Akt and eNOS, phosphorylation. The PI3K inhibitor and a dominant-negative mutant of Akt suppressed Akt and eNOS phosphorylation and NO production. Our results demonstrated that FKN stimulated angiogenesis by activating the Raf-1/MEK/ERK and PI3K/Akt/eNOS/NO signal pathways via the G protein-coupled receptor CX3CR1, indicating that two pathways are required for full angiogenic activity of FKN. This study suggests that FKN may play an important role in the pathophysiological process of inflammatory angiogenesis.

scholarly journals Real-Time Detection of Interactions between the Human Oxytocin Receptor and G Protein-Coupled Receptor Kinase-2

2004 ◽  
Vol 18 (5) ◽  
pp. 1277-1286 ◽  
Author(s):  
Ahmed Hasbi ◽  
Dominic Devost ◽  
Stéphane A. Laporte ◽  
Hans H. Zingg
Keyword(s):  
Real Time ◽  
G Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Oxytocin Receptor ◽  
Dominant Negative ◽  
Receptor Kinase ◽  
G Protein Coupled Receptor ◽  
Time Resolved ◽  
G Protein Coupled

Abstract Although the oxytocin receptor (OTR) mediates many important functions including uterine contractions, milk ejection, and maternal behavior, the mechanisms controlling agonist-induced OTR desensitization have remained unclear, and attempts to demonstrate involvement of a G protein-coupled receptor kinase (GRK) have so far failed. Using the OTR as a model, we demonstrate here directly for the first time the dynamics of agonist-induced interactions of a GRK with a G protein-coupled receptor in real time, using time-resolved bioluminescence resonance energy transfer. GRK2/receptor interactions started within 4 sec, peaked at 10 sec, and decreased to less than 40% within 8 min. By contrast, β-arrestin/OTR interactions initiated only at 10 sec, reached plateau levels at 120 sec, but remained stable with little decrease thereafter. Physical GRK2/OTR association was further demonstrated by coimmunoprecipitation of endogenous GRK2 with activated OTR. In COS-7 cells, which express low levels of GRK2 and β-arrestin, overexpression of GRK2 and β-arrestin increased receptor phosphorylation, desensitization, and internalization to the high levels observed in human embryonic kidney 293 cells. By contrast, specific inhibition of endogenous GRK2 by dominant-negative mutants robustly inhibited OTR phosphorylation and internalization as well as arrestin/OTR interactions. These data characterize the temporal and causal relationship of GRK-2/OTR and β-arrestin/OTR interactions and establish GRK/OTR interaction as a prerequisite for β-arrestin-mediated OTR desensitization.

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