Interactions of phosducin with defined G protein beta gamma-subunits

Phosducin has recently been identified as a cytosolic protein that interacts with the beta gamma-subunits of G proteins and thereby may regulate transmembrane signaling. It is expressed predominantly in the retina but also in many other tissues, which raises the question of its potential specificity for retinal versus nonretinal beta gamma-subunits. We have therefore expressed and purified different combinations of beta- and gamma-subunits from Sf9 cells and have also purified transducin-beta gamma from bovine retina and a mixture of beta gamma complexes from bovine brain. Their interactions with phosducin were determined in a variety of assays for beta gamma function: support of ADP-ribosylation of alpha(0) by pertussis toxin, enhancement of the GTPase activity of alpha(0), and enhancement of rhodopsin phosphorylation by the beta-adrenergic receptor kinase 1 (beta ARK1). There were only moderate differences in the effects of the various beta gamma complexes alone on alpha(0), but there were marked differences in their ability to support beta ARK1 catalyzed rhodopsin phosphorylation. Phosducin inhibited all beta gamma-mediated effects and showed little specificity toward specific defined beta gamma complexes with the exception of transducin-beta gamma (beta(1) gamma(1)), which was inhibited more efficiently than the other beta gamma combinations. In a direct binding assay, there was no apparent selectivity of phosducin for any beta gamma combination tested. Thus, in contrast to beta ARK1, phosducin does not appear to discriminate strongly between different G protein beta- and gamma-subunits.