Article,

Identification of a C-terminal binding site for G-protein betagamma-subunits in phosducin-like protein

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FEBS Lett, 401 (2-3): 243-6 (January 1997)Schroder, S Bluml, K Dees, C Lohse, M J Research Support, Non-U.S. Gov't Netherlands FEBS letters FEBS Lett. 1997 Jan 20;401(2-3):243-6..

Abstract

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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