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| TR |
TITLE |
AUTHORS |
KEYWORDS |
MATERIALS & METHODS
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MICROSCOPIC
TECHNIQUES |
SPECIES |
MORPHOLOGY |
CELL
LINE |
| 631 |
Differential
Kinetic and Spatial Patterns of Arrestin and G Protein-mediated ERK Activation by the Angiotensin
II Receptor |
Seungkirl
Ahn, Sudha K. Shenoy, Huijun Wei, and Robert J. Lefkowitz |
angiotensin
II type 1A
receptor, Gprotein, B-arrest in2, nuclear translocation, cytoplasmic endosomal
vesicles |
One day after transfection, cells
were divided into collagen-coated 35-mm glass bottom dishes (MatTek, Ashland, MA)
for confocal microscopy.
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confocal
microscopy |
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HEK-293 |
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The seven-membrane-spanning angiotensin
II type 1A receptor activates the mitogen-activated protein kinases extracellular signal-regulated
kinases 1 and 2 (ERK1/2) by distinct pathways dependent on either G protein (likely Gq/G11)
or -arrestin2. Here we sought to distinguish the kinetic and spatial patterns that characterize
ERK1/2 activated by these two mechanisms. We utilized -arrestin RNA interference, the protein
kinase C inhibitor Ro-31-8425, a mutant angiotensin II receptor (DRY/AAY), and a mutant angiotensin
II peptide (SIIangiotensin), which are incapable of activating G proteins, to isolate the two
pathways in HEK-293 cells. G protein-dependent activation was rapid (peak <2 min), quite
transient (t1/2 2 min), and led to nuclear translocation of the activated ERK1/2 as assessed
by confocal microscopy. In contrast, -arrestin2-dependent activation was slower (peak 5–10
min), quite persistent with little decrement noted out to 90 min, and entirely confined to the
cytoplasm. Moreover, ERK1/2 activated via -arrestin2 accumulated in a pool of cytoplasmic endosomal
vesicles that also contained the internalized receptors and -arrestin. Such differential regulation
of the temporal and spatial patterns of ERK1/2 activation via these two pathways strongly implies
the existence of distinct physiological endpoints. |
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