A switch 3 point mutation in the alpha subunit of transducin yields a unique dominant-negative inhibitor.
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 (alphaT) subunits. Here we have described a novel dominant-negative mutation, made in the background of a chimera consisting of alphaT and the alpha subunit of G(i1) (designated alphaT*), 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 alphaT* to undergo rhodopsin-stimulated GDP-GTP exchange, whereas substituting glutamic acid for arginine at this position yielded an alphaT* subunit (alphaT*(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 alphaT*, alphaT*(R238A) and alphaT*(R238K), the alphaT*(R238E) mutant did not respond to aluminum fluoride (AlF4(-)), as read out by changes in Trp-207 fluorescence. However, surprisingly, we found that alphaT*(R238E) effectively blocked rhodopsin-catalyzed GDP-GTP exchange on alphaT*, as well as rhodopsin-stimulated phosphodiesterase activity. Analysis by high pressure liquid chromatography indicated that the alphaT*(R238E) mutant exists in a nucleotide-free state. Nucleotide-free forms of G alpha subunits were typically very sensitive to proteolytic degradation, but alphaT*(R238E) exhibited a resistance to trypsin-proteolysis similar to that observed with activated forms of alphaT*. 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.
Pereira
Ryan
R
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, USA.
Cerione
Richard A
RA
D017607:Aluminum Compounds / Q000737:chemistry / Q000494:pharmacology; D000818:Animals; D001120:Arginine / Q000737:chemistry; D002384:Catalysis; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D006152:Cyclic GMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005459:Fluorides / Q000737:chemistry / Q000494:pharmacology; D005799:Genes, Dominant; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000737:chemistry; D006160:Guanosine Triphosphate / Q000737:chemistry; D008954:Models, Biological; D008958:Models, Molecular; D009154:Mutation; D009711:Nucleotides / Q000737:chemistry; D017354:Point Mutation; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012160:Retina / Q000378:metabolism; D012243:Rhodopsin / Q000737:chemistry; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D015293:Transducin / Q000737:chemistry* / Q000235:genetics*; D014357:Trypsin / Q000737:chemistry / Q000494:pharmacology
D017607:Aluminum Compounds; D009711:Nucleotides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D012243:Rhodopsin; D001120:Arginine; D014357:Trypsin; D015293:Transducin; D006152:Cyclic GMP; D005459:Fluorides; C032311:aluminum fluoride