Dominant negative mutants of transducin-alpha that block activated receptor.
Mutations counterpart to dominant negative RasSer17Asn in the alpha-subunits of heterotrimeric G-proteins are known to also produce dominant negative effects. The mechanism of these mutations remains poorly understood. Here, we examined the effects and mechanism of the Ser43Cys and Ser43Asn mutants of transducin-like chimeric Gtalpha* in the visual signaling system. Our analysis showed that both mutants have reduced affinity for GDP and are likely to exist in an empty-or partially occupied-pocket state. S43C and S43N retained the ability to interact with Gtbetagamma and, as heterotrimeric proteins, bind to photoexcited rhodopsin (R*). The interaction with R* is unproductive as the mutants failed to bind GTPgammaS and become activated. S43C and S43N inhibited R*-dependent activation of Gtalpha* and Gtalpha, apparently by blocking R*. Finally, both Gtalpha* mutants lacked interaction with the gamma-subunit of PDE6, an effector protein in phototransduction. These results indicate that the S43C and S43N mutants of Gtalpha* are dominant negative inhibitors that bind and block the activated receptor in a mechanism that parallels that of RasSer17Asn. Dominant negative mutants of Gtalpha sequestering R*, such as S43C and S43N, may become useful instruments in probing the mechanisms of visual dysfunctions caused by abnormal phototransduction signaling.
Natochin
Michael
M
Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
Barren
Brandy
B
Artemyev
Nikolai O
NO
D000244:Adenosine Diphosphate / Q000378:metabolism; D002384:Catalysis; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009711:Nucleotides / Q000378:metabolism; D037342:Pertussis Toxin / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011485:Protein Binding; D021122:Protein Subunits / Q000378:metabolism; D012243:Rhodopsin / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015293:Transducin / Q000235:genetics* / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D009711:Nucleotides; D021122:Protein Subunits; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000244:Adenosine Diphosphate; D012243:Rhodopsin; D037342:Pertussis Toxin; D010727:Phosphoric Diester Hydrolases; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D014357:Trypsin; D015293:Transducin