April 2010
Volume 51, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2010
657WTAPELL663 is a SignatureTransduction Motif in Ca2+-Sensor Modulation of ROS-GC1
Author Affiliations & Notes
  • T. Duda
    Regulatory & Molecular Biology, Salus University, Elkins Park, Pennsylvania
  • R. K. Sharma
    Regulatory & Molecular Biology, Salus University, Elkins Park, Pennsylvania
  • Footnotes
    Commercial Relationships  T. Duda, None; R.K. Sharma, None.
  • Footnotes
    Support  HL084584 (TD) DC005349 (RKS)
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 1097. doi:https://doi.org/
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      T. Duda, R. K. Sharma; 657WTAPELL663 is a SignatureTransduction Motif in Ca2+-Sensor Modulation of ROS-GC1. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1097. doi: https://doi.org/.

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

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Abstract

Purpose: : To determine the universal mechanism of ROS-GC1 activation by Ca2+-sensors.

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

Results: : Molecular modelling studies show that in all membrane guanylate cyclases including the phototransduction-linked ROS-GC1 the kinase homology domain has a bilobal shape with a large cleft at the interface of the two lobes. Within the predominantly alpha-helical larger lobe there is a conserved hydrophobic motif 657WTAPELL663. Initially, in the prototype model of ANF receptor guanylate cyclase this motif was predicted to be involved (directly or indirectly) in activation of the cyclase catalytic domain. In search for a universal model of the activation mechanism of membrane guanylate cyclase this prediction was experimentally validated for GCAPs- and S100B-dependent activation of ROS-GC1. 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 only minimal response to both GCAPs and S100B. Alanine scanning of the motif resulted in Ala mutants that exhibited varying responses to the tested Ca2+ sensors. Within the WTAPELL motif the W657 residue was of the dominant significance.

Conclusions: : Like in the hormone peptide receptor ANF-RGC model the 657WTAPELL663 motif is critical for the GCAP- and S100B modulated Ca2+ signalling of ROS-GC1.Support: NIH awards HL 084584 (TD), DC 005349 (RKS)

Keywords: calcium • photoreceptors 
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