April 2009
Volume 50, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2009
Novel GCAP1-Modulated Transduction Motif of ROS-GC1
Author Affiliations & Notes
  • T. M. Duda
    Regulatory & Molecular Biology, Salus University, Elkins Park, Pennsylvania
  • R. K. Sharma
    Regulatory & Molecular Biology, Salus University, Elkins Park, Pennsylvania
  • Footnotes
    Commercial Relationships  T.M. Duda, None; R.K. Sharma, None.
  • Footnotes
    Support  NIH awards DC 005349 (RKS), HL 084584 (TD)
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 2995. doi:https://doi.org/
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    • Get Citation

      T. M. Duda, R. K. Sharma; Novel GCAP1-Modulated Transduction Motif of ROS-GC1. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2995. doi: https://doi.org/.

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

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Abstract

Purpose: : To determine the mechanism through which GCAP1 signals activation of the ROS-GC1 catalytic domain.

Methods: : Molecular modelling together with biochemical techniques involving point- and deletion-mutagenesis, guanylate cyclase assays and Western blots were used.

Results: : Through homology based modelling a conserved hydrophobic motif 657WTAPELL663 within the ROS-GC1 kinase-like domain was predicted to be involved (directly or indirectly) in the GCAP1-dependent activation of the cyclase catalytic domain. This prediction was experimentally validated. Deletion of the entire 657WTAPELL663 motif resulted in a mutant that had almost identical basal activity and Km for the substrate GTP as the wild-type ROS-GC1; the mutant, however, had significantly (70-80%) lowered response to GCAP1 stimulation. Mutation to alanine of the individual residues of the motif resulted in respective Ala mutants that exhibited varying responses to GCAP1 stimulation. This indicates that each amino acid within the 657WTAPELL663 motif contributes selectively to the motif’s function. The W657 residue of the motif plays the dominant role in the motif’s activity, however. Its mutation to alanine results in 70% lower stimulation as compared with the wild type ROS-GC1.

Conclusions: : The ROS-GC1 657WTAPELL663 motif is pivotal for transduction of the GCAP1 binding signal to the cyclase catalytic domain. Within this motif W657 is the key residue. Upon GCAP1 binding there is a reorientation of the W657 side chain that pushes the remainder of the motif, 658TAPELL663, to the surface. As a consequence, hydrophobic 658TAPELL663 gets exposed. This exposure makes these residues accessible for interaction with another transduction module of ROS-GC1, possibly with the catalytic domain, propagation of the GCAP1 binding signal, and ultimately activation of the catalytic module.

Keywords: signal transduction • photoreceptors • protein structure/function 
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