A Switch 3 Point Mutation in the α Subunit of Transducin Yields a Unique Dominant-negative Inhibitor

Abstract The rhodopsin/transducin-coupled vertebrate vision system has served as a paradigm for G protein-coupled signaling. We have taken advantage of this system to identify new types of constitutively active, transducin-α (αT) subunits. Here we have described a novel dominant-negative mutation, made in the background of a chimera consisting of αT and the α subunit of Gi1 (designated αT*), which involves the substitution of a conserved arginine residue in the conformationally sensitive Switch 3 region. Changing Arg-238 to either lysine or alanine had little or no effect on the ability of αT* to undergo rhodopsin-stimulated GDP-GTP exchange, whereas substituting glutamic acid for arginine at this position yielded an αT* subunit (αT*(R238E)) that was incapable of undergoing rhodopsin-dependent nucleotide exchange and was unable to bind or stimulate the target/effector enzyme (cyclic GMP phosphodiesterase). Moreover, unlike the GDP-bound forms of αT*, αT*(R238A) and αT*(R238K), the αT*(R238E) mutant did not respond to aluminum fluoride (), as read out by changes in Trp-207 fluorescence. However, surprisingly, we found that αT*(R238E) effectively blocked rhodopsin-catalyzed GDP-GTP exchange on αT*, as well as rhodopsin-stimulated phosphodiesterase activity. Analysis by high pressure liquid chromatography indicated that the αT*(R238E) mutant exists in a nucleotide-free state. Nucleotide-free forms of Gα subunits were typically very sensitive to proteolytic degradation, but αT*(R238E) exhibited a resistance to trypsin-proteolysis similar to that observed with activated forms of αT*. Overall, these findings indicated that by mutating a single residue in Switch 3, it is possible to generate a unique type of dominant-negative Gα subunit that can effectively block signaling by G protein-coupled receptors.