Development of a novel DnaE intein-based assay for quantitative analysis of G-protein-coupled receptor internalization

2011 ◽  
Vol 417 (1) ◽  
pp. 65-72 ◽  
Author(s):  
Yaping Zhang ◽  
Wen Yang ◽  
Linjie Chen ◽  
Ying Shi ◽  
Guo Li ◽  
...  
Keyword(s):  
Quantitative Analysis ◽  
G Protein ◽  
Receptor Internalization ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled

scholarly journals A pH-Sensitive Fluor, CypHerTM 5, Used to Monitor Agonist-Induced G Protein-Coupled Receptor Internalization in Live Cells

BioTechniques ◽  
2002 ◽  
Vol 33 (5) ◽  
pp. 1152-1157 ◽  
Author(s):  
E.J. Adie ◽  
S. Kalinka ◽  
L. Smith ◽  
M.J. Francis ◽  
A. Marenghi ◽  
...  
Keyword(s):  
G Protein ◽  
Ph Sensitive ◽  
Receptor Internalization ◽  
Live Cells ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled

scholarly journals Quantitative Analysis of the Bidirectional Viral G-Protein-Coupled Receptor and Lytic Latency-Associated Nuclear Antigen Promoter of Kaposi's Sarcoma-Associated Herpesvirus

2012 ◽  
Vol 86 (18) ◽  
pp. 9683-9695 ◽  
Author(s):  
Isaac B. Hilton ◽  
Dirk P. Dittmer
Keyword(s):  
Quantitative Analysis ◽  
G Protein ◽  
Binding Sites ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Nuclear Antigen ◽  
G Protein Coupled Receptor ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
G Protein Coupled

Kaposi's sarcoma-associated herpesvirus (KSHV) establishes sustained latent persistence in susceptible cells. This is dependent on the latency-associated nuclear antigen (LANA). Understanding how LANA transcription is regulated thus aids our fundamental understanding of KSHV biology. Two hundred ninety-four base pairs are sufficient to regulate LANA transcription in response to the viral RTA protein and RBPjκ. The same region controls K14/viral G-protein-coupled receptor (vGPCR) transcription in the opposite direction. We used a quantitative analysis in conjunction with specific nucleotide substitutions and defined gain-of-function and loss-of-function RTA mutants to dissect this region. We used a bidirectional reporter driving red and green luciferase to study the LANApi and K14p promoters simultaneously. This established that LANApi/K14p functions as a canonical bidirectional promoter. Both were TATA dependent. K14p was favored by ∼50-fold in this context. Eliminating the distal LANApi TATA box increased maximal output and lowered the induction threshold (T) of K14p even further. Two RBPjκ binding sites were independently required; however, at high concentrations of RTA, direct interactions with an RTA-responsive element (RRE) could complement the loss of one RBPjκ binding site. Intracellular Notch (ICN) was no longer able to activate RBPjκ in the viral context. This suggests a model whereby KSHV alters ICN-RBPjκ gene regulation. When the architecture of this pair of head-to-head RBPjκ binding sites is changed, the sites now respond exclusively to the viral transactivator RTA and no longer to the host mediator ICN.

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 Dynamin and β-Arrestin Reveal Distinct Mechanisms for G Protein-coupled Receptor Internalization

1996 ◽  
Vol 271 (31) ◽  
pp. 18302-18305 ◽  
Author(s):  
Jie Zhang ◽  
Stephen S. G. Ferguson ◽  
Larry S. Barak ◽  
Luc Ménard ◽  
Marc G. Caron
Keyword(s):  
G Protein ◽  
Receptor Internalization ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled

Role of beta-Arrestin in Mediating Agonist-Promoted G Protein-Coupled Receptor Internalization

Science ◽  
1996 ◽  
Vol 271 (5247) ◽  
pp. 363-366 ◽  
Author(s):  
S. S. G. Ferguson ◽  
W. E. Downey ◽  
A.-M. Colapietro ◽  
L. S. Barak ◽  
L. M nard ◽  
...  
Keyword(s):  
G Protein ◽  
Receptor Internalization ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled
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