Quantification of the tissue levels and function of the G-protein regulator phosducin-like protein (PhlP)

Phosducin-like protein is a protein with widespread expression that has been shown to be capable of inhibiting G-protein function in vitro. However, it is nor clear whether it is expressed in sufficient amounts to actually exert such functions in vivo. Here we quantify the expression of the short and the long splice variants of phosducin-like protein, PhlP(s) and PhlP(l). Western blots of various rat tissues showed that PhlP(l) was by far the dominant splice variant; its levels were 1.5-2 pmol/mg cytosolic protein in brain, liver and kidney, and about 0.5 pmol/mg cytosolic protein in lung, heart and skeletal muscle. These values correspond to concentrations of 150-200 nM and 50 nM, respectively. The levels of PhlP(s) were about 20-fold lower. Recombinant phosducin, PhlP(l) and PhlP(s) inhibited the interaction between G-protein alpha- und beta gamma -subunits with IC50-values of 6 nM, 6 nM and 90 nM, respectively, as determined by G(beta gamma)-dependent ADP-ribosylation of G alpha (il) by pertussis-toxin. Thus, tissue concentrations of PhlP(l) are clearly sufficient to affect G-protein function in vivo, while the expression levels and the G(beta gamma)-affinity of PhlP(s) are most likely too low to have significant inhibitory effects on G(beta gamma) (G-protein beta gamma -subunits).