April 2010
Volume 51, Issue 13
ARVO Annual Meeting Abstract  |   April 2010
N-Myristoylation in GCAP1 Affects Its Ca(2+) Sensitivity and Interaction With RetGC1
Author Affiliations & Notes
  • I. V. Peshenko
    Pennsylvania Coll. of Optometry, Salus University, Elkins Park, Pennsylvania
  • A. M. Dizhoor
    Pennsylvania Coll. of Optometry, Salus University, Elkins Park, Pennsylvania
  • Footnotes
    Commercial Relationships  I.V. Peshenko, None; A.M. Dizhoor, None.
  • Footnotes
    Support  EY11522, The Pennsylvania Lions Sight Conservation and Eye Research Foundation.
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 1091. doi:
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      I. V. Peshenko, A. M. Dizhoor; N-Myristoylation in GCAP1 Affects Its Ca(2+) Sensitivity and Interaction With RetGC1. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1091.

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

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Purpose: : Guanylyl cyclase activating proteins (GCAPs) are myristoylated Ca(2+)/Mg(2+)-binding proteins that impart Ca(2+) sensitivity to retinal guanylyl cyclase (RetGC). The purpose of this study was to elucidate the functional role of the N-myrisloyl group in the interactions between GCAP1 and RetGC1.

Methods: : Myristoylated and non-myristoylated GCAP1, either untagged or GFP-tagged, was produced in E. coli and HEK293 cells and its metal binding properties and the ability to bind and activate recombinant RetGC1 in vitro or in co-transfected HEK293 cells were assessed using methods previously described in [1, 2].

Results: : When myristoylation was blocked by expressing the wild type GCAP1 in the absence of N-myristoyl transferase or by introducing a G2A mutation, it demonstrated Ca(2+)- and Mg(2+)-dependent changes in Trp fluorescence similar to myristoylated WT GCAP1. However, while the spectral changes reflecting transition from an apo-form to a RetGC1 activator form were unaffected in the non-myristoylated GCAP1, the Ca(2+) sensitivity of the transition from the RetGC activator into a RetGC inhibitor form was shifted toward higher Ca(2+) concentration range. Consequently, there was also a decrease in Ca(2+)-dependent inhibition of RetGC1 by the non-myristoylated GCAP1. The lack of myrisloylation also had two additional effects on RetGC1 regulation: it decreased the apparent affinities of both Ca(2+)- and Mg(2+)-bound GCAP1 for RetGC1 and reduced the maximal stimulation of RetGC1 by Mg(2+)-bound GCAP1 in vitro. However, GFP-tagged G2A GCAP1 was still able to associate with RetGC1 in HEK293 cells, although less efficiently than myristoylated GFP-tagged WT GCAP1.

Conclusions: : The presence of N-myristoyl group in GCAP1 contributes to all three key elements of the cyclase regulation: binding to RetGC, its maximal activation, and the Ca(2+) sensitivity of its inhibition.

References: : [1] Peshenko & Dizhoor (2006) J. Biol. Chem. 281, 23830-41; [2] Peshenko et al. (2008) J. Biol. Chem. 283, 21747-57.

Keywords: protein structure/function • calcium • microscopy: confocal/tunneling 

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