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.

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In vertebrate rod cells, the activated alpha-subunit of rod transducin interacts with the gamma (regulatory) subunits of phosphodiesterase to disinhibit the catalytic subunits. A 22-amino acid long region of rod transducin involved in phosphodiesterase activation has recently been identified. We have used peptides from this region of rod transducin and from several other G protein alpha-subunits to study the nature and specificity of the G protein alpha-effector interaction. Although peptides derived from rod transducin, cone transducin and gustducin are similar, only the rod peptide is capable of activating rod phosphodiesterase. Using substituted peptides we have identified five residues on one exposed face of rod transducin as important to phosphodiesterase activation. These results disagree with previous models which propose that loop regions of rod transducin interact with phosphodiesterase gamma.

Site-directed mutagenesis was used to identify residues responsible for the > 1,000-fold difference in ouabain sensitivity between the rat Na,K-ATPase al and at2 isoforms. A series of mutagenized cDNAs was constructed that replaced residues of the rat at2 subunit with the corresponding residues from the rat al subunit. These cDNAs were cloned into a mammalian episomal expression vector (EBOpLPP) and expressed in ouabainsensitive primate cells. Either of two single substitutions introduced into the rat ai2 subunit cDNA (Leu-111-*Arg or Asn-122-*Asp) conferred partial resistance (-10 ,uM ouabain) upon transformed cells. This resistance was intermediate between the levels conferred by the rat al cDNA (-500 ,uM ouabain) and the rat a2 cDNA (-0.2 ,iM ouabain). A double substitution of the rat a2 cDNA (Leu-111--*Arg and Asn-122-Asp) conferred a resistance level equivalent to that obtained with rat al. These results demonstrate that the residues responsible for isoform-specific differences in ouabain sensitivity are located at the ends of the H1-H2 extracellular domain. The combination of site-directed mutagenesis and episomal expression provides a useful system for the selection and analysis of mutants.