The rod and cone transducins are specific G proteins originally thought to be present only in photoreceptor cells of the vertebrate retina. Transducins convert light stimulation of photoreceptor opsins into activation of cyclic GMP phosphodiesterase (reviewed in refs. 5-7). A transducin-like G protein, gustducin, has been identified and cloned from rat taste cells. We report here that rod transducin is also present in vertebrate taste cells, where it specifically activates a phosphodiesterase isolated from taste tissue. Furthermore, the bitter compound denatonium in the presence of taste-cell membranes activates transducin but not Gi. A peptide that competitively inhibits rhodopsin activation of transducin also blocks taste-cell membrane activation of transducin, arguing for the involvement of a seven-transmembrane-helix G-protein-coupled receptor. These results suggest that rod transducin transduces bitter taste by coupling taste receptor(s) to taste-cell phosphodiesterase. Phosphodieterase-mediated degradation of cyclic nucleotides may lead to taste-cell depolarization through the recently identified cyclic-nucleotide-suppressible conductance.

Guanine nucleotide-binding regulatory protein (G protein) by dimers that were active in reconstitution assays were produced in insect cells using the baculovirus/Sf`9 insect cell expression system. Sf9 cells were infected either singly or in combination with recombinant baculoviruses containing a human G-protein .31 gene or a bovine G-protein 72 gene. It was possible to express the .i1 and 72 gene products independently of each other in this system, as determined by using immunological and metabolic labeling techniques. Further, the ability of recombinant fi and/or y chains to function in dermed biochemical assays of fy activity was assessed for membrane extracts and supernatant fractions from infected Sf9 cells. Extracts of cells expressing fi or y chain alone were inactive in these assays, whereas those from cells coinfected with fil and 72 did display activity. These assays were used to identify recombinant By dimer migration during chromatographic purification, and the recombinant dimers were purified to near homogeneity. Both the membrane-associated and soluble By dimers facilitated rhodopsin-catalyzed guanosine 5'-[ythioltriphosphate binding to Gta, the GTP-binding subunit of the retinal G protein transducin (KO.5 of 13 ± 2 and 36 ± 5 nM, respectively). Both recombinant fBy dimers also facilitated the pertussis toxin-catalyzed ADP-ribosylation of Gta with equal potency (Ko.5 of 9 ± 1 and 10 ± 3 nM for membrane and soluble dimers, respectively). [3HIMevalono-lactone labeling showed that the 72 subunits of membraneassociated fBy dimers incorporated radiolabel, whereas in the soluble form they did not. Thus, prenyl modification of 72 directs the membrane association of the fi172 dimer and increases its apparent affinity for receptor, but it is not required for the functional interaction(s) of the dimer.Guanine nucleotide-binding regulatory proteins (G proteins) are a family of proteins that serve as vital links in the chain of proteins that transduce extracellular signals to diverse intracellular effectors, such as adenylyl cyclases, phosphodiesterases, and phospholipases, and to ion channels in the membrane (1-3). All signal-transducing G proteins identified to date are membrane-associated heterotrimers composed of subunits referred to as a, 3, and 'y. The (3 and y subunits of G proteins do not dissociate under physiological conditions and thus behave essentially as a monomer; in addition, fBy dimers occur in almost all tissues as a complex mixture oftwo 3proteins (4), each bound to as many as three y subunits (5, 6). Gf3'y displays a number of in vitro activities related to the guanine nucleotide binding and activation of a subunits, including reversal of aluminum fluoride-activated Gsa (ref. 7; G., stimulatory G protein), inhibition or reversal ofguanosine 5'-[y-thio]triphosphate (GTP[yS]) binding to Ga (8,9), and enhancement of pertussis and cholera toxin catalyzed ADPribosylation of Ga subunits (10, 11). Gfy is required for efficient coupling of Ga subunits to cell surface receptors (12-15). Fina...