Interaction of phosducin-like protein with G protein betagamma subunits.
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.
Thibault
C
C
Ernest Gallo Clinic and Research Center, Department of Neurology, University of California at San Francisco, San Francisco, California 94110, USA.
Sganga
M W
MW
Miles
M F
MF
D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D019556:COS Cells; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D009419:Nerve Tissue Proteins; C098789:PDCL protein, human; D019204:GTP-Binding Proteins