May 2006
Volume 47, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2006
Characterization of Candidate Dominant–Negative Mutants of Transducin–
Author Affiliations & Notes
  • N.O. Artemyev
    Physiology, Univ, Iowa, IA
  • B. Barren
    Physiology, Univ, Iowa, IA
  • M. Natochin
    Physiology, Univ, Iowa, IA
  • Footnotes
    Commercial Relationships  N.O. Artemyev, None; B. Barren, None; M. Natochin, None.
  • Footnotes
    Support  NIH Grant EY12682
Investigative Ophthalmology & Visual Science May 2006, Vol.47, 3729. doi:
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      N.O. Artemyev, B. Barren, M. Natochin; Characterization of Candidate Dominant–Negative Mutants of Transducin– . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3729.

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Abstract

Purpose: : To investigate potential dominant–negative mutants of transducin–α and identify the mechanism of their action.

Methods: : The S43N, S43C and R238E substitutions were introduced into a tranducin–like chimeric Gtα*–subunit. The nucleotide–bound state of the mutants was assessed using the trypsin–protection assay and HPLC chromatography. The interaction of mutant Gtα* with Gtßγ was tested with pertussis toxin–catalyzed ADP–ribosylaion. The ability of mutants to bind photoexcited rhodopsin (R*), and undergo R*–stimulated guanine nucleotide exchange was examined using ROS–pull down and GTPγS–binding assays, respectively. The mutant GTPase activity was characterized in the steady–state and single turnover measurements of GTP hydrolysis. A binding assay utilizing the fluorescently–labeled γ–subunit of PDE6 (Pγ) was employed to monitor the effector function.

Results: : Transducin mutant Gtα*R238E exists in a nucleotide–bound state, binds Gßγ and is fully capable of activational coupling to R*. This mutation results in a significant impairment of Gtα*’s ability to hydrolyze GTP and interact with the inhibitory subunit of PDE6. S43N and S43C displayed severely diminished affinity for GDP while retaining an interaction with Gtßγ. Heterotrimeric S43N and S43C bind R*, but fail to bind GTP and become activated. Furthermore, S43N and S43C in the presence of Gtßγ potently inhibited R*–dependent activation of transducin.

Conclusions: : The phenotype of R238E is entirely inconsistent with that of a dominant–negative inhibitor as recently reported [1]. In contrast, S43N and S43C are potent dominant–negative inhibitors of transducin signaling. The mechanism of these mutants involves sequestration of R*. 1. Pereira R., Cerione RA. (2005) J Biol Chem 280:35696–35703.

Keywords: photoreceptors • signal transduction 
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