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| TR |
TITLE |
AUTHORS |
KEYWORDS |
MATERIALS & METHODS
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MICROSCOPIC
TECHNIQUES |
SPECIES |
MORPHOLOGY |
CELL
LINE |
| 581 |
Gi
and G subunits both define selectivity of G protein activation by 2-adrenergic receptors |
Scott
K. Gibson and Alfred G. Gilman |
fluorescence
resonance energy transfer, GTP-binding protein, subunits, GTP-binding protein, subunits, small
interfering RNA |
Five hours after transfection,
cells were split into four 35-mm glass bottom culture dishes (MatTek).
|
Fluorescence
Microscopy |
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|
HeLa |
| Abstract |
| |
Previous studies of the specificity of receptor interactions
with G protein subunits in living cells have relied on measurements of second messengers or
other downstream responses. We have examined the selectivity of interactions between 2-adrenergic
receptors ( 2R) and various combinations of Gi and G subunit isoforms by measuring changes in
FRET between Gi–yellow fluorescent protein and cyan fluorescent protein–G chimeras
in HeLa cells. All combinations of Gi1, -2, or -3 with G1, -2, or -4 were activated to some
degree by endogenous 2Rs as judged by agonist-dependent decreases in FRET. The degree of G
protein activation is determined by the combination of Gi and G subunits rather than by the
identity of an individual subunit. RT-PCR analysis and small interfering RNA knockdown of 2R
subtypes, followed by quantification of radiolabeled antagonist binding, demonstrated that HeLa
cells express 2a- and 2b-adrenergic receptor isoforms in a 2:1 ratio. Increasing receptor
number by overexpression of the 2aR subtype minimized the differences among coupling preferences
for Gi and G isoforms. The molecular properties of each Gi, G, and 2-adrenergic receptor
subtype influence signaling efficiency for the 2-adrenergic receptormediated signaling pathway. |
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