Phosducin is a ubiquitous G-protein regulator.

Abstract

Phosducin is a 33-kDa cytosolic regulator of G-protein-mediated signaling that has previously been thought to be specific for retina and pineal gland. In this study, we show widespread tissue distribution of phosducin by the amplification of its cDNA and the detection of two different transcripts in Northern analyses in liver, lung, heart, brain, and retina. On the protein level, phosducin could be detected in 12 bovine tissues by immune precipitation and subsequent Western analysis using anti-phosducin antibodies generated in two different species. Masking of phosducin in direct Western blots appears to explain the failure to detect phosducin in earlier studies. The concentration of phosducin in bovine brain was calculated in the range of 10 pmol/mg total cytosolic protein (-1I,uM), whereas in the other tissues, it was slightly less. In these concentrations, phosducin inhibited receptor-stimulated adenylyl cyclase activity in cell membranes by about 50%. Taken together, our results indicate that phosducin is a ubiquitous regulator of G-protein function.GTP-binding proteins (G proteins) couple many kinds of cell surface receptors to intracellular effectors or ion channels and are thereby crucial for the specificity, velocity, and amplification of signal transduction across the cell membrane (1, 2). Agonist-occupied receptors stimulate the binding of GTP to the a subunits of G proteins (Ga), which, in turn, promotes the dissociation of the G protein 13y (Gf3y) subunits, allowing both the Ga and GfBy subunits to exert their various specific functions (3). These transduction cascades are regulated by multiple mechanisms, which have been investigated most thoroughly for the j3-adrenergic receptor/stimulatory G protein (Gs)/adenylyl cyclase (4) and the rhodopsin/transducin (Gt)/cGMP phosphodiesterase (5) systems. Regulation occurs predominantly at the receptor level but also at the level of G proteins themselves.Phosducin is a 33-kDa soluble phosphoprotein present in the retina (6, 7) and the developmentally related pineal gland (8, 9). It forms complexes with the P3'y subunits of transducin, the retinal G protein (6). The Gf3y binding site has been mapped to its N terminus (10, 11). Phosducin affects G-proteinmediated signal transduction by high-affinity binding to Gf3y and may, thereby, regulate the number of f3y subunits available to interact with the Ga subunits. Phosducin has been shown to inhibit the intrinsic GTPase activity of Gs, and inhibitory and "other" G proteins (G1 and Go, respectively) equally well (12). Similarly, it has been shown to reduce the activity of Gt (13 This study was undertaken to test this hypothesis and to investigate the presence of phosducin and its mRNA in nonretinal/pineal tissues. We demonstrate that it occurs in a wide variety of bovine tissues at the mRNA level and protein level, respectively, and that its concentration in these tissues is sufficient to act as a G-protein regulator. MATERIALS AND METHODSRNA Isolation and Northern Blot Analysis. Total RNA was isolated from...

Phosphorylation of Phosducin and Phosducin-like Protein by G Protein-coupled Receptor Kinase 2

“…Several lines of evidence suggest that membrane-bound GRK2 activity could be enhanced by stimulation of endogenous heterotrimeric G proteins with aluminum fluoride (22). Because phosducin has been described as an inhibitor of G␤␥-stimulated GRK2 activity toward agonist-activated receptors by competing for binding with free G␤␥ subunits (31,34), purified bovine phosducin was included in our assays to test the participation of endogenous G␤␥ subunits in the observed stimulation of GRK2 activity. As expected, phosducin (600 nM) did inhibit light-dependent phosphorylation of rhodopsin (2 M) by GRK2 (Fig.…”

“…Through interaction with free G␤␥ subunits, phosducin and PhLP have been shown to interfere with a variety of GPCR signaling pathways triggered by different G proteins (25,29,31,32,42). The reported modulation of phosducin function by PKA phosphorylation provides a clear feedback regulatory mechanism in the case of receptor signaling leading to changes in cellular cAMP concentrations.…”

“…On the other hand, phosducin and phosducin-like proteins (PhLP) are able to preferentially bind to G␤␥ subunits, thus interfering with G␤␥-mediated signaling pathways and with re-association with G␣⅐GDP (24 -30). Phosducin is a ϳ33-kDa soluble phosphoprotein that was first identified in the retina (24) and later shown to be ubiquitously expressed (25,31). The homologous PhLP also shows a broad pattern of expression (28,29).…”

Rethinking the role of phosducin: Light-regulated binding of phosducin to 14-3-3 in rod inner segments

“…The putative expression of trace amounts of Pd in other parts of the nervous system, and indeed in non-neuronal cell types remains controversial (21)(22)(23). First isolated as a phosphoprotein from dark-adapted rat retinal extracts (24), Pd was found to gradually dephosphorylate on light exposure.…”

Phosducin interacts with the G-protein βγ-dimer of ciliate protozoanBlepharisma japonicumupon illumination

“…The interactions of both 14-3-3 protein and G␤␥ with Pdc are dependent on the phosphorylation state of Pdc. The discovery of Pdc's role in retinal photoreceptor cells of mammals and the identification of a similar downregulation function for phosducin-like protein, such as PhLP1, in other tissues and lower eukaryotic cells (Lee et al, 1987;Reig et al, 1990;Danner and Lohse, 1996;Flanary et al, 2000;Kasahara et al, 2000;Blaauw et al, 2003) indicate that these highly conserved proteins, which belong to subgroup I of the phosducin protein family, play an important role as cytosolic regulators of G-protein functions. However, lately there is growing evidence that PhLP may also have other cellular functions.…”