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A Rhodopsin Transport Assay by High-Content Imaging Analysis
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A Rhodopsin Transport Assay by High-Content Imaging Analysis

Published on: January 16, 2019

Rhodopsin recognition by mutant G(s)alpha containing C-terminal residues of transducin

M Natochin1, K G Muradov, R L McEntaffer

  • 1Department of Physiology, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.

The Journal of Biological Chemistry
|January 25, 2000
PubMed

Insights

Specific C-terminal residues of transducin alpha (G(t)alpha) are crucial for its activation by light-activated rhodopsin (R*). These residues, particularly Cys347 and Gly348, mediate G(t)alpha/R* coupling and influence nucleotide exchange.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cellular Signaling

Background:

  • Heterotrimeric G protein alpha-subunits are key regulators of cellular signaling.
  • The C-terminal regions of G protein alpha-subunits are critical for receptor-mediated activation.
  • Transducin alpha (G(t)alpha) is involved in visual signal transduction.

Purpose of the Study:

  • To identify critical determinants in the C-terminus of G(t)alpha responsible for activation by light-activated rhodopsin (R*).
  • To investigate the role of specific C-terminal residues and the switch II region in G(t)alpha activation and nucleotide exchange.

Main Methods:

  • Construction and analysis of chimeric G(s)alpha/G(t)alpha proteins with C-terminal substitutions.
  • Assays to measure binding of mutant G(s)alpha to R*.
  • Measurement of intrinsic nucleotide exchange rates in mutant G(s)alpha proteins.

Main Results:

  • A chimeric protein with 11 C-terminal residues of G(t)alpha efficiently bound and was activated by R*.
  • Cys347 and Gly348 were essential for G(t)alpha activation by R*, while Asp346 played a modest role.
  • Mutations in the switch II region of G(s)alpha, mimicking G(t)alpha, reduced nucleotide exchange rates.

Conclusions:

  • The C-terminal residues of G(t)alpha, specifically Cys347 and Gly348, are essential for its coupling and activation by R*.
  • Communication between the G(t)alpha C-terminus and switch II region is not essential for R*-mediated activation but may regulate intrinsic nucleotide exchange.
  • These findings elucidate mechanisms of G protein activation and regulation by receptors.

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