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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 Gbetagamma 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 Gbetagamma dimer in vitro and in vivo. Using PhLP glutathione S-transferase fusion proteins, we show that PhLP directly binds Gbetagamma in vitro. Studies with a series of truncated PhLP fusion proteins indicate independent binding of Gbetagamma to both the amino- and C-terminal halves of PhLP. Protein-protein interactions between Gbetagamma and PhLP are inhibited by the alpha subunit of Go and Gi3, suggesting that PhLP can bind only free Gbetagamma. Finally, we show that PhLP complexes, at least partially, with Gbetagamma in vivo. Following overexpression of epitope-tagged PhLP together with Gbeta1gamma2 proteins in COS-7 cells, a PhLP-Gbetagamma complex is co-immunoprecipitated by monoclonal antibody directed against the epitope tag. Similarly, polyclonal anti-PhLP antibody co-precipitates endogenous PhLP and Gbetagamma proteins from NG108-15 cell lysates. These data are consistent with the hypothesis that PhLP is a widely expressed modulator of Gbetagamma function. Furthermore, because alternate forms of the PhLP transcript are expressed, there may be functional implications for the existence of two Gbetagamma-binding domains on PhLP.
Highlights
The G␣ subunit binds a molecule of GDP
Based on sequence homology with phosducin, we have suggested that phosducinlike protein (PhLP) proteins regulate G␥ signaling in nonretinal tissues
To determine whether PhLP directly interacts with G␥, we generated PhLP and PhLP short (PhLPS) glutathione S-transferase (GST) fusion proteins and examined their ability to bind G␥ purified from bovine brain
Summary
The G␣ subunit binds a molecule of GDP. Upon ligand binding, the receptor catalyzes the exchange of GDP for GTP on G␣ that causes its activation and dissociation from the tightly bound G␥ complex.1 Inactivation and reassociation of the heterotrimer is initiated by the hydrolysis of bound GTP into GDP by an intrinsic GTPase activity of the G␣ subunit. In a typical binding assay, following immobilization on glutathione-agarose beads, fusion proteins at a final concentration of 0.5–1.0 M were incubated with 50 –100 nM G␥ purified from bovine brain in 50 l of phosphate-buffered saline (PBS) containing 0.01% Lubrol for 2 h at 4 °C. To determine whether PhLP directly interacts with G␥, we generated PhLP and PhLPS GST fusion proteins and examined their ability to bind G␥ purified from bovine brain.
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