April 2009
Volume 50, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2009
Interactions of GCAP1 and RetGC1: The Stoichiometry of Binding in Live Transfected Cells
Author Affiliations & Notes
  • A. M. Dizhoor
    Pennsylvania College of Optometry, Salus University, Elkins Park, Pennsylvania
  • E. V. Olshevskaya
    Pennsylvania College of Optometry, Salus University, Elkins Park, Pennsylvania
  • I. V. Peshenko
    Pennsylvania College of Optometry, Salus University, Elkins Park, Pennsylvania
  • Footnotes
    Commercial Relationships  A.M. Dizhoor, None; E.V. Olshevskaya, None; I.V. Peshenko, None.
  • Footnotes
    Support  NIH grant EY11522, Pennsylvania Lions Sight Conservation and Eye Research Foundation.
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 5455. doi:
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      A. M. Dizhoor, E. V. Olshevskaya, I. V. Peshenko; Interactions of GCAP1 and RetGC1: The Stoichiometry of Binding in Live Transfected Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5455.

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

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Abstract

Purpose: : Ca2+/Mg2+ binding proteins, GCAPs, regulate photoreceptor guanylyl cyclase (retGC1 and 2) in vertebrate photoreceptors. The structure of the GCAP/retGC complexes remains poorly understood, including the stoichiometry of their binding. The purpose of this study was to evaluate the GCAP1:retGC1 complex stoichiometry using fluorescently tagged functional proteins co-expressed in cultured cells.

Methods: : Recombinant GCAP1 and retGC1 fluorescently tagged with eGFP and monomeric dsRed, respectively, were co-expressed in HEK 293 cells as described previously [1], and the ratio between the two fluorescent tags co-localized in the membranes determined using confocal microscopy was compared against a "1:1" chimera standard, dsRedRetGC1-GCAP1eGFP, expressed as a single fusion protein in the same experiments.

Results: : When expressed in HEK293 cells, GCAP1 and retGC1 were co-localized in the membranes, thus creating a characteristic pattern strikingly different from the diffuse pattern of GCAP1 expressed in the absence of retGC1 [1] or in the presence of retGC1 mutants insensitive to GCAP1. The co-localization required the presence of the intracellular segment of the cyclase, but not its catalytic activity: deletion of the catalytic center did not prevent binding of GCAP1. With the increased levels of GCAP1 versus retGC1 expression, the ratio of the eGFP:dsRed fluorescence for the GCAP1:retGC1 complex in the membranes approached that of the "1:1" chimera standard. The maximal GCAP1:retGC1 ratio in the complex calculated using a fractional saturation function was ~1.05.

Conclusions: : The results indicate that each retGC1 molecule can directly bind with high affinity only one GCAP1 molecule.

Keywords: signal transduction • photoreceptors • protein structure/function 
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