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

Abstract

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 not 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 α- und βγ-subunits with IC₅₀-values of 6 nM, 6 nM and 90 nM, respectively, as determined by G_βγ-dependent ADP-ribosylation of Gα_i1 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_βγ-affinity of PhlP_s are most likely too low to have significant inhibitory effects on G_βγ (G-protein βγ-subunits).