Interaction of phosducin-like protein with G protein βγ subunits

Abstract

Phosducin-like protein (PhLP), a widely expressed ethanol-responsive gene (Miles, M. F., Barhite, S., Sganga, M., and Elliott, M. (1993) Proc. Natl. Acad. Sci U.S. A. 90, 10831-10835), is a homologue of phosducin, a known major regulator of Gβγ signaling in retina and pineal gland. However, although phosducin has a well characterized role in retinal phototransduction, function of the PhLP remains unclear. In this study we examine the ability of PhLP to bind Gβγ dimer in vitro and in vivo. Using PhLP glutathione S-transferase fusion proteins, we show that PhLP directly binds Gβγ in vitro. Studies with a series of truncated PhLP fusion proteins indicate independent binding of Gβγ, to both the amino- and C-terminal halves of PhLP. Protein-protein interactions between Gβγ and PhLP are inhibited by the α subunit of G 0 and G 13, suggesting that PhLP can bind only free Gβγ. Finally, we show that PhLP complexes, at least partially, with Gβγ in vivo. Following overexpression of epitope-tagged PhLP together with Gβ 1γ 2 proteins in COS-7 cells, a PhLP-Gβγ complex is co- immunoprecipitated by monoclonal antibody directed against the epitope tag. Similarly, polyclonal anti-PhLP antibody co-precipitates endogenous PhLP and Gβγ proteins from NG108-15 cell lysates. These data are consistent with the hypothesis that PhLP is a widely expressed modulator of Gβγ function. Furthermore, because alternate forms of the PhLP transcript are expressed, there may be functional implications for the existence of two Gβγ-binding domains on PhLP.