Retrovirally Mediated Transfer of a G Protein-coupled Receptor Kinase (GRK) Dominant-negative Mutant Enhances Endogenous Calcitonin Receptor Signaling in Chinese Hamster Ovary Cells

An important aspect of the image analysis of immunocytochemical preparations is the evaluation of colocalization of different molecules. The aim of the present study is to introduce image analysis methods to identify double-labeled locations exhibiting the highest association of two fluorophores and to characterize their pattern of distribution. These methods will be applied to the analysis of the cotrafficking of adenosine A2A and dopamine D2 receptors belonging to the G protein–coupled receptor family and visualized by means of fluorescence immunocytochemistry in Chinese hamster ovary cells after agonist treatment. The present procedures for colocalization have the great advantage that they are, to a large extent, insensitive to the need for a balanced staining with the two fluorophores. Thus, these procedures involve image processing, visualization, and analysis of colocalized events, using a covariance method and a multiply method and the evaluation of the identified colocalization patterns. Moreover, the covariance method offers the possibility of detecting and quantitatively characterizing anticorrelated patterns of intensities, whereas the immediate detection of colocalized clusters with a high concentration of labeling is a possibility offered by the multiply method. The present methods offer a new and sensitive approach to detecting and quantitatively characterizing strongly associated fluorescence events, such as those generated by receptor–receptor interaction, and their distribution patterns in dual-color confocal laser microscopy.

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Development of a Cyclic Adenosine Monophosphate Assay for Gi-Coupled G Protein-Coupled Receptors by Utilizing the Endogenous Calcitonin Activity in Chinese Hamster Ovary Cells

Assay and Drug Development Technologies ◽

10.1089/adt.2010.0361 ◽

2011 ◽

Vol 9 (5) ◽

pp. 522-531 ◽

Cited By ~ 5

Author(s):

Yuren Wang ◽

Yan Kong ◽

Gan Ju Shei ◽

Liya Kang ◽

Mary Ellen Cvijic

Keyword(s):

G Protein ◽

Chinese Hamster Ovary ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

G Protein Coupled Receptors ◽

Ovary Cells ◽

G Protein Coupled

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Control of the Cardiac Muscarinic K+Channel by β-Arrestin 2

Journal of Biological Chemistry ◽

10.1074/jbc.m011007200 ◽

2001 ◽

Vol 276 (15) ◽

pp. 11691-11697 ◽

Cited By ~ 7

Author(s):

Z. Shui ◽

I. A. Khan ◽

T. Haga ◽

J. L. Benovic ◽

M. R. Boyett

Keyword(s):

Muscarinic Receptor ◽

Chinese Hamster Ovary ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

K Channel ◽

Receptor Kinase ◽

Ovary Cells ◽

M2 Muscarinic Receptor ◽

Receptor Channel ◽

In Cells

Control of the cardiac muscarinic K+current (iK,ACh) by β-arrestin 2 has been studied. In Chinese hamster ovary cells transfected with m2 muscarinic receptor, muscarinic K+channel, receptor kinase (GRK2), and β-arrestin 2, desensitization of iK,AChduring a 3-min application of 10 μmACh was significantly increased as compared with that in cells transfected with receptor, channel, and GRK2 only (fade in current increased from 45 to 78%). The effect of β-arrestin 2 was lost if cells were not co-transfected with GRK2. Resensitization (recovery from desensitization) of iK,AChin cells transfected with β-arrestin 2 was significantly slowed (time constant increased from 34 to 232 s). Activation and deactivation of iK,AChon application and wash-off of ACh in cells transfected with β-arrestin 2 were significantly slowed from 0.9 to 3.1 s (time to half peak iK,ACh) and from 6.2 to 13.8 s (time to half-deactivation), respectively. In cells transfected with a constitutively active β-arrestin 2 mutant, desensitization occurred in the absence of agonist (peak current significantly decreased from 0.4 ± 0.05 to 0.1 ± 0.01 nA). We conclude that β-arrestin 2 has the potential to play a major role in desensitization and other aspects of the functioning of the muscarinic K+channel.

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