Аннотация
Phosducin-like protein (PhLP) has recently been identified as a ubiquitous
inhibitor of G-protein betagamma-subunit (G betagamma)-mediated signaling,
with an affinity about 5-fold lower than that of phosducin. The G
betagamma binding site of phosducin has been suggested to be contained
in its N-terminus. A region corresponding to this N-terminus is lacking
in PhLP, suggesting that PhLP must utilize a different mode of G
betagamma binding. To map the G betagamma binding site in PhLP, a
series of deletion mutants were constructed, expressed in E. coli
as glutathione S-transferase (GST) fusion proteins, and the purified
fusion proteins were examined for their ability to attenuate G(o)
GTPase activity. Progressive N-terminal truncations of PhLP caused
only minor reductions in potency, whereas the complementary N-terminal
PhLP fragments turned out to be inactive. We further identified a
short C-terminal segment comprising residues 168 to 195 that inhibited
G0 GTPase activity similar in efficacy and potency to full-length
PhLP. This C-terminal fragment was also capable of antagonizing a
second G betagamma-mediated function, the enhancement of rhodopsin
phosphorylation by the beta-adrenergic receptor kinase. Taken together,
these data indicate that PhLP interacts with G betagamma via a short
C-terminal binding site which is distinct from that identified previously
in phosducin.
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