The same mutation in Gsalpha and transducin alpha reveals behavioral differences between these highly homologous G protein alpha-subunits.
Mutating Arg-238 to Glu (R238E) in the switch 3 region of a transducin alpha (*Talpha) in which 27 aa of the GTPase domain have been replaced with those of the alpha-subunit of the inhibitory G protein 1 (Gi1alpha), was reported to create an alpha-subunit that is resistant to activation by GTPgammaS, is devoid of resident nucleotide, and has dominant negative (DN) properties. In an attempt to create a DN stimultory G protein alpha (Gsalpha) with a single mutation we created Gsalpha-R265E, equivalent to *Talpha-R238E. Gsalpha-R265E has facilitated activation by GTPgammaS, a slightly facilitated activation by GTP but much reduced receptor plus GTP stimulated activation, and an apparently unaltered ability to interact with receptor as seen in ligand binding studies. Further, the activity profile of Gsalpha-R265E is that of an alpha-subunit with unaltered or increased GTPase activity. The only change in Gsalpha that is similar to that in *Talpha is that the apparent affinity for guanine nucleotides is decreased in both proteins. The molecular basis of the changed properties are discussed based on the known crystal structure of Gsalpha and the changes introduced by the same mutation in a *Talpha (Gtalpha*) with only 23 aa from Gi1alpha. Gtalpha*-R238E, with four fewer mutations in switch 3, was reported to show no evidence of DN properties, is activated by GTPgammaS, and has reduced GTPase activity. The data highlight a critical role for the switch 3 region in setting overall properties of signal-transducing GTPases.
Zurita
Adolfo R
AR
Laboratory of Neurobiology, Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
Birnbaumer
Lutz
L
D058666:Adrenergic beta-2 Receptor Agonists; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002460:Cell Line; D002864:Chromogranins; D004789:Enzyme Activation / Q000187:drug effects; D044385:GTP-Binding Protein alpha Subunits / Q000235:genetics / Q000378:metabolism*; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006728:Hormones / Q000494:pharmacology; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D017434:Protein Structure, Tertiary; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D015293:Transducin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D058666:Adrenergic beta-2 Receptor Agonists; D002864:Chromogranins; D044385:GTP-Binding Protein alpha Subunits; D006728:Hormones; D018343:Receptors, Adrenergic, beta-2; D006160:Guanosine Triphosphate; C406732:GNAS protein, human; D019205:GTP-Binding Protein alpha Subunits, Gs; D015293:Transducin