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

Phosducin-like protein (PhLP) has recently been identified as a ubiquitous inhibitor of G-protein beta gamma-subunit (G(beta gamma))-mediated signaling, with an affinity about 5-fold lower than that of phosducin. The G(beta gamma) 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(beta gamma) binding, To map the G(beta gamma) 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 G(o) GTPase activity similar in efficacy and potency to full-length PhLP, This C-terminal fragment was also capable of antagonizing a second G(beta gamma)-mediated function, the enhancement of rhodopsin phosphorylation by the beta-adrenergic receptor kinase. Taken together, these data indicate that PhLP interacts with G(beta gamma) via a short C-terminal binding site which is distinct from that identified previously in phosducin.