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R.K. Sharma, K.W. Koch, C. Lange, T. Duda; Selective GCAP1 Binding Site in ROS–GC1 . Invest. Ophthalmol. Vis. Sci. 2006;47(13):804.
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ROS–GC1 is a photoreceptor membrane guanylate cyclase, which through two Ca2+ sensor proteins, GCAP1 and GCAP2, controls phototransduction. GCAP1 and GCAP2 modulated domains in ROS–GC1 are distinct. In this investigation the biochemical perimeters of the GCAP1–modulated site have been defined.
Mutagenesis/expression/reconstitution experiments, peptide competition, real–time binding (surface plasmon resonance spectroscopy) experiments, co–immunoprecipitation and cross–linking studies were carried out.
Deletion and point mutation/expression studies have identified two GCAP1 regulatory sites within the juxtamembrane portion of the kinase homology domain of ROS–GC1, M445–L456 and L503–I522. Co–immunoprecipitation and SPR experiments show that the ROS–GC1 soluble fragment aa S470 to A653 directly binds GCAP1 (KD value of 0.25 µM as determined by SPR) but not GCAP2. The binding occurs in the presence and absence of Ca2+ with comparable KD values. Although both peptides, M445–L456 and L503–I522, inhibit GCAP1–dependent ROS–GC1 activity only the L503–I522 peptide binds GCAP1.
The ROS–GC1–GCAP1 signaling pathway operates via a specific site on ROS–GC1; the site is distinct from the GCAP2–modulatory site, and is Ca2+–independent.
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