期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 280, 期 42, 页码 35696-35703出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M504935200
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资金
- NEI NIH HHS [EY06429] Funding Source: Medline
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- alpha(alpha T) subunits. Here we have described a novel dominant- negative mutation, made in the background of a chimera consisting of alpha T and the alpha subunit of G(i1) ( designated alpha T*), 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 alpha T* to undergo rhodopsin- stimulated GDP- GTP exchange, whereas substituting glutamic acid for arginine at this position yielded an alpha T* subunit ( alpha T*( 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 alpha T*, alpha T*( R238A) and alpha T*( R238K), the alpha T*( R238E) mutant did not respond to aluminum fluoride ( AlF4-), as read out by changes in Trp- 207 fluorescence. However, surprisingly, we found that alpha T*( R238E) effectively blocked rhodopsin- catalyzed GDP- GTPexchange on alpha T*, as well as rhodopsin- stimulated phosphodiesterase activity. Analysis by high pressure liquid chromatography indicated that the alpha T*( R238E) mutant exists in a nucleotide- free state. Nucleotide- free forms of G alpha subunits were typically very sensitive to proteolytic degradation, but alpha T*( R238E) exhibited a resistance to trypsin- proteolysis similar to that observed with activated forms of alpha T*. Overall, these findings indicated that by mutating a single residue in Switch 3, it is possible to generate a unique type of dominant- negative G alpha subunit that can effectively block signaling by G protein- coupled receptors.
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