May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Evidence for a Direct Protein-Protein Interaction Between Rod Transducin and Retinal Guanylate Cyclase
Author Affiliations & Notes
  • D. H. Rosenzweig
    Pharmacology, University of Miami, Miami, Florida
  • K. Levay
    Pharmacology, University of Miami, Miami, Florida
  • K. S. Nair
    Pharmacology, University of Miami, Miami, Florida
  • I. V. Peshenko
    Hafter Laboratories, Pennsylvania College of Optometry, Elkins Park, Pennsylvania
  • A. M. Dizhoor
    Hafter Laboratories, Pennsylvania College of Optometry, Elkins Park, Pennsylvania
  • J. W. Crabb
    Opthalmic Research, Cole Eye Institute Cleavland Clinic, Cleveland, Ohio
  • V. Z. Slepak
    Pharmacology, University of Miami, Miami, Florida
  • Footnotes
    Commercial Relationships  D.H. Rosenzweig, None; K. Levay, None; K.S. Nair, None; I.V. Peshenko, None; A.M. Dizhoor, None; J.W. Crabb, None; V.Z. Slepak, None.
  • Footnotes
    Support  NIH GM60019 (VZS), AHA predoctoral fellowship 0515233B (DHR)
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 1674. doi:https://doi.org/
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      D. H. Rosenzweig, K. Levay, K. S. Nair, I. V. Peshenko, A. M. Dizhoor, J. W. Crabb, V. Z. Slepak; Evidence for a Direct Protein-Protein Interaction Between Rod Transducin and Retinal Guanylate Cyclase. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1674. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : Illumination of vertebrate rod photoreceptors causes compartmental re-localization of several signaling proteins including the trimeric G protein transducin. We have previously shown that relocalization of rod transducin (Gt) is mediated by interaction-restricted diffusion (Rosenzweig et. al., 2007). Here we tested for novel binding partners of transducin which could directly influence its diffusion and localization.

Methods: : Rod Gαt was specifically immunoprecipitated from isolated bovine and murine rod outer segments (ROS). The antigenic peptide for Rod Gαt antibodies was used as a control. Specifically bound proteins were resolved by SDS-PAGE followed by silver staining. Bound proteins were excised, subjected to trypsin digestion, analyzed by mass spectrometry and confirmed by western blot. Reconstitution experiments were performed by transient transfection of Cos-7 cells with mammalian plasmid vectors containing Gαt and retinal guanylate cyclase (retGC). Guanylate cyclase activity was measured using radioactive GTP, and radioactive cGMP was used as a control. The domain on retGC to which Gαt interacts was identified by pull-down using GST-fusion proteins of the kinase homology and catalytic domains (KHD & CD) of retGC.

Results: : Mass spectrometry analysis of protein bands specifically bound to rod Gαt reliably identified retGC as a binding partner. Western blot analysis of rod transducin immunoprecipitation from bovine ROS, mouse ROS, and transfected Cos-7 cells confirmed this interaction in vivo and in vitro. Gαt specifically bound to the GST fusion kinase homology domain of retGC, and to a much lesser extent, to the GST-catalytic domain. The GDP-bound form of Gαt associated stronger than the GTPγS-bound form. Purified rod Gαt and Gβγ subunits had no effect on retGC-mediated cGMP synthesis.

Conclusions: : Rod transducin can bind specifically to retinal guanylate cyclase in vertebrate photoreceptors. The interaction occurs primarily at the kinase homology domain of retGC and does not alter the cylase activity.

Keywords: photoreceptors • protein structure/function • retina 
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