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 ␤...

Phosphorylation of G protein-coupled receptors by ß-adrenergic receptor kinases (ßARK) requires the presence of G protein ßy subunits. We have investigated the ability of the two ßARK isoforms to distinguish between defined recombinant ßy subunits. ßARK2 had an about 25% lower specific activity than ßARKl towards rhodopsin and the ß2-adrenergic receptor but the two kinases shared the selectivity for ßy subunits: ßy complexes consisting of ßi or ß2 in combination with Y2, ys, and ¥i were more efficacious than those with y3 or ßiyi. Thus, while ßARKs differentiate between defined ßy subunits, ßy complexes do not discriminate between ßARK isoforms.

Arrestins are cytosolic proteins involved in the desensitization of G-protein-coupled receptors. We report the cloning of trout red blood cell arrestin which shows 76, 82 and 52% identity with bovine beta-arrestin1, beta-arrestin2 and retinal arrestin respectively. Antibodies were generated against the C-terminus of trout red blood cell arrestin. These antibodies detected arrestin in erythrocyte cytosol and were able to precipitate the native protein. The Na+/H+ antiporter of trout red blood cell is activated by beta-adrenergic stimulation and is then desensitized whereas the transmembrane signalling pathway is not. To investigate the subcellular distribution of arrestin on beta-adrenergic activation and desensitization of the antiporter, precipitation experiments were carried out on trout erythrocytes. A desensitization-dependent shift in cytosolic arrestin to the membranes could not be detected using the immunoprecipitation technique but we cannot exclude the possibility that a small number of cytosolic arrestins might be involved in the regulation of membrane proteins in trout erythrocyte. Recombinant trout arrestin was produced in a protease-deficient Escherichia coli strain and its functionality was tested in a reconstituted rhodopsin assay. The recombinant protein provides a suitable tool for investigating the target for arrestin in trout red blood cell, which still remains to be identified.