Interactions of Phosducin with Defined G Protein βγ-Subunits

Abstract

Phosducin has recently been identified as a cytosolic protein that interacts with the ␤␥-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 ␤␥-subunits. We have therefore expressed and purified different combinations of ␤-and ␥-subunits from Sf9 cells and have also purified transducin-␤␥ from bovine retina and a mixture of ␤␥ complexes from bovine brain. Their interactions with phosducin were determined in a variety of assays for ␤␥ function: support of ADP-ribosylation of ␣ o by pertussis toxin, enhancement of the GTPase activity of ␣ o , and enhancement of rhodopsin phosphorylation by the ␤-adrenergic receptor kinase 1 (␤ARK1). There were only moderate differences in the effects of the various ␤␥ complexes alone on ␣ o , but there were marked differences in their ability to support ␤ARK1 catalyzed rhodopsin phosphorylation. Phosducin inhibited all ␤␥-mediated effects and showed little specificity toward specific defined ␤␥ complexes with the exception of transducin-␤␥ (␤ 1 ␥ 1 ), which was inhibited more efficiently than the other ␤␥ combinations. In a direct binding assay, there was no apparent selectivity of phosducin for any ␤␥ combination tested. Thus, in contrast to ␤ARK1, phosducin does not appear to discriminate strongly between different G protein ␤-and ␥-subunits.Guanine nucleotide-binding proteins (G proteins) are transducers between heptahelical receptors and various effectors. Traditionally, G proteins have been classified according to their ␣-subunits, but in the last years a plethora of effects have been assigned to the ␤␥-subunits. Thus, they have been shown to regulate adenylyl cyclases, phospholipases C-␤ and A 2 , PI3-kinase, the ADP-ribosylation factor and several ion channels (for reviews, see Refs. 1 and 2). In addition, ␤␥-subunits provide a membrane attachment site for the ␤-adrenergic receptor kinase (␤ARK) 1 which allows translocation of the kinase from the cytosol to the cell membrane and thus in close proximity to its substrate receptors (3, 4). Phosphorylation of G protein-coupled receptors by ␤ARK is thought to represent the first step of homologous desensitization; it is followed by binding of proteins of the arrestin family which results in uncoupling of receptors and their G proteins (5, 6).Another cytosolic protein which interacts with G protein ␤␥-dimers is phosducin, a phosphoprotein originally purified from bovine retina as a complex with the ␤␥-subunits of G t (7). By binding to G protein ␤␥-subunits phosducin can inhibit G protein-mediated signaling (8, 9). Furthermore, phosducin can compete with ␤ARK for the ␤␥-subunits and can thereby impair ␤ARK-mediated phosphorylation of receptors (10). The interaction of phosducin with G proteins is markedly reduced following its phosphorylation by protein kinase A (8, 11).The possibility of a common motif for the interaction of all these proteins with ␤...

The Role of Electrostatic Interactions in the Regulation of the Membrane Association of G Protein βγ Heterodimers

“…More recent studies show that phosducin is ubiquitously expressed (46) and that it interacts equally well with other ␤␥ isoforms (47). As shown in Fig.…”

Conditional Stimulation of Type V and VI Adenylyl Cyclases by G Protein βγ Subunits

“…1, C and D, the nonisoprenylated G␤ 1 ␥ 2 (C68S) did not further stimulate G␣ s or forskolin-stimulated hACV or hACVI activity. Moreover, when phosducin, which binds and sequesters G␤␥ (39), was added, the ability of G␤ 1 ␥ 2 to stimulate hACV or hACVI activities was obliterated (Fig. 1E).…”

“…Sf9 membranes containing the desired AC forms were preincubated at room temperature for 2 min with G␤ 1 ␥ 2 or G␤ 1 ␥ 2 C68S at the indicated concentrations before initiating the reactions. The AC assays were performed as described before (38,39) in a total volume of 100 l in the presence of 5 mM Mg 2ϩ . The reactions were initiated by the addition of G␣ s or forskolin at the indicated concentrations and continued at room temperature for 15 min.…”

Effects of two Gβγ-binding proteins – N-terminally truncated phosducin and β-adrenergic receptor kinase C terminus (βARKct) – in heart failure

“…15 The Gbgbinding affinities of bARKct (B300 nmol/l), phosducin and an N-terminally truncated variant ('nt-del-phosducin' nt-del-phd) (15 and 33 nmol/l) are comparable. [15][16][17] In contrast to bARKct, however, phosducin and nt-delphosducin do not cause overall resensitization of the badrenergic receptor system owing to simultaneous inhibition of GDP release from Ga subunits. 11,14,18 The structure of phosducin and nt-del-phd are compared schematically in Figure 1.…”