Quantification of the tissue levels and function of the G-protein regulator phosducin-like protein (PhlP).
Phosducin-like protein is a protein with wide-spread 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, PhlPs and PhlP1. Western blots of various rat tissues showed that PhlP1 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 PhlPs were about 20-fold lower. Recombinant phosducin, PhlP1 and PhlPs inhibited the interaction between G-protein alpha- und betagamma-subunits with IC50-values of 6 nM, 6 nM and 90 nM, respectively, as determined by Gbetagamma-dependent ADP-ribosylation of Galphai1 by pertussis-toxin. Thus, tissue concentrations of PhlP1 are clearly sufficient to affect G-protein function in vivo, while the expression levels and the Gbetagamma-affinity of PhlPs are most likely too low to have significant inhibitory effects on Gbetagamma (G-protein betagamma-subunits).
Schröder
S
S
Institute of Pharmacology, University of Würzburg, Germany.
Lohse
M J
MJ
D000818:Animals; D002352:Carrier Proteins / Q000032:analysis* / Q000502:physiology; D019204:GTP-Binding Proteins / Q000502:physiology*; D018832:Molecular Chaperones; D009419:Nerve Tissue Proteins / Q000032:analysis* / Q000502:physiology; D021122:Protein Subunits; D051381:Rats
D002352:Carrier Proteins; D018832:Molecular Chaperones; D009419:Nerve Tissue Proteins; C498320:Pdcl protein, rat; D021122:Protein Subunits; D019204:GTP-Binding Proteins